development of an upscaled bio-artificial kidney
The development of cell based bioartificial kidney device (BAK) could improve existing dialysis therapies for the removal of protein-bound toxins. A key requirement for the BAK is the formation of a “living membrane” consisting of a tight renal cells monolayer with preserved functional organic ion transporters, on an artificial porous membrane.
The main aim of this thesis is to develop an upscaled BAK prototype, making use of conditionally immortalized human proximal tubule epithelial cells (ciPTEC), seeded on functionalized hollow fiber membranes (HFM). The aim and general structure of this thesis are presented in chapter 1.
Chapter 2 introduces the background of the kidney anatomy and function. It gives an overview of the current renal replacement therapies (RRT), their advantages and limitations. After a short history review of the BAK, the requirements for this concept are presented. In fact, we highlight that there is a strong need (i) for a reliable and consistent cell line, (ii) for HFM with tailored transport and surface properties, (iii) but also for device related logistics such as cost-effective manufacturing, storage, and distribution process.
Chapter 3 presents the upscaling of a “living membrane” for a BAK device. First, the development of a double L-Dopa and collagen IV coating on the external surface of a commercially available hollow fiber membrane (HFM) module is reported and the HFM transport properties are studied. Secondly, Organic Cationic Transporter (OCT)-expressing ciPTEC are seeded on the functionalized HFM and the quality of the obtained monolayer is studied. Immunochemistry results and reduced inulin-FITC transport prove that this living membrane, developed under static culturing conditions, exhibits a uniform, reproducible and tight ciPTEC monolayer, on the outside surface of the HFM. Finally, our work shows that ciPTEC cultured in this upscaled system feature active OCT, crucial for the removal of uremic cationic metabolites.
Chapter 4 investigates strategies for achieving a good quality OCT2-expressing ciPTEC monolayer on the inside surface of the polymeric HFM. This configuration could be preferred for the development of a clinically relevant BAK due to a better preservation of the cell monolayer against friction and to a greater similarity with the natural proximal tubule. We first optimize the functionalization of the internal surface of the polymeric fiber to achieve high transport of metabolites. Secondly, we investigate several cell seeding parameters in order to achieve a tight ciPTEC monolayer on the inside surface of the HFM. The successful seeding parameters for the formation of the tight barrier are identified. In comparison to other studies, the procedure developed here presents two advantages: a single cell infusion and a short proliferation time.
Chapter 5 investigates the ability of alternative flat surfaces to support the formation of a “living membrane”. Following our hypothesis, not only collagen IV but also other elements of the natural kidney epithelial extra cellular matrix can be used to coat membranes to support ciPTEC. Moreover, we investigate the ability of a positively charged polymer membrane to support the adhesion of the negatively charged cells. Preliminary results for PES membranes coated with HS, as well as for PEI membranes are presented. Both surfaces improve the ciPTEC adhesion and, when combined with the application of L-Dopa, lead to the formation of a monolayer. The transepithelial transport tests indicate a barrier function of the monolayer as well as the function of the OCT2 on PES coated with L-Dopa/HS. Cell attachment strength experiments indicate good cell-cell interaction for both PES membranes coated with L-Dopa/CIV and for PEI membranes. As a result, both studied surfaces – (i) PES coated with L-Dopa/HS and (ii) PEI membranes - could be good candidates for membranes for BAK applications.
Chapter 6 presents the successful upscaling of a “living membrane” of organic anionic transporter 1 (OAT1)-expressing ciPTEC on functionalized MicroPES HFM. The uniform and tight ciPTEC monolayer, achieved on the external side of the HFM, demonstrates active OAT, crucial for the removal of uremic anionic toxins, such as indoxyl sulfate (IS). Furthermore, we study the immune response of the ciPTEC. The ciPTEC are fully polarized: the pro-inflammatory cytokines are mainly released towards the apical - dialysate - compartment, which would not be in direct contact with the patient body fluid. This would greatly reduce risks associated with eventual pro-inflammatory and immunogenic effects of the ciPTEC.
Finally chapter 7 presents the general conclusions and reflections on the future directions in the development of a clinically relevant BAK.