Co-axial Printing of Convoluted Proximal Tubule for Kidney Disease Modeling

Despite the increasing incidence of kidney-related diseases, we are still far from understanding the underlying mechanisms of these diseases and their progression. This lack of understanding is partly because of a poor replication of the diseasesin vitro,limited to planar culture. Advancing towards three-dimensional models, hereby we propose coaxial printing to obtain microfibers containing a helical hollow microchannel. These recapitulate the architecture of the proximal tubule (PT), an important nephron segment often affected in kidney disorders. A stable gelatin/alginate-based ink was formulated to allow printability while maintaining structural properties. Fine-tuning of the composition, printing temperature and extrusion rate allowed for optimal ink viscosity that led to coiling of the microfiber's inner channel. The printed microfibers exhibited prolonged structural stability (42 days) and cytocompatibility in culture. Healthy conditionally immortalized PT epithelial cells and a knockout cell model for cystinosis (CTNS-/-) were seeded to mimic two genotypes of PT. Upon culturing for 14 days, engineered PT showed homogenous cytoskeleton organization as indicated by staining for filamentous actin, barrier-formation and polarization with apical markerα-tubulin and basolateral marker Na+/K+-ATPase. Cell viability was slightly decreased upon prolonged culturing for 14 days, which was more pronounced inCTNS-/-microfibers. Finally,CTNS-/-cells showed reduced apical transport activity in the microfibers compared to healthy PT epithelial cells when looking at breast cancer resistance protein and multidrug resistance-associated protein 4. Engineered PT incorporated in a custom-designed microfluidic chip allowed to assess leak-tightness of the epithelium, which appeared less tight inCTNS-/-PT compared to healthy PT, in agreement with itsin vivophenotype. While we are still on the verge of patient-oriented medicine, this system holds great promise for further research in establishing advancedin vitrodisease models

Engineering the (vascularized) Proximal Tubule, a Guide Towards Functionality

This thesis focused on the fabrication of kidney proximal tubule models. A coaxial printing system allowed the robust and straightforward fabrication of coiled perfusable microfibers, replicating the kidney proximal convoluted tubules. The formed alginate-gelatin microfibers offer a complex tubular shape in which the cells exhibit mature markers, such as functional transporters and polarized monolayers. Moreover, this model has proven to support both healthy and cystinotic proximal tubule cell lines and allows for mechanistically studying tubulopathies. Though distinctive 3D-printing strategies offer opportunities to mimic the structure of the convoluted proximal tubule, printing resolution remains restricted for soft hydrogel materials. Therefore, a unique method was studied, whereby post-printing treatment induced resolution enhancement of methacrylated hyaluronic acid / gelatin methacryloyl (HAMA/GelMA) based hydrogels. This shrinking technique opens a wide application to create higher resolutions for 3D printing. However, we also found that the polycationic shrinking reagents used were relatively toxic for our cells of interest. Therefore, more research is needed to prevent the direct exposure of cells to the shrinking reagents. Using melt electrowriting, scaffolds were fabricated that are self-supportive, yet small-sized and highly porous. These scaffolds enable direct access to the basolateral and luminal cell sides to facilitate solute exchange with vasculature in immediate proximity, which is critical for functional proximal tubule constructs. The polymer applied, polycaprolactone, is used to prepare biomaterials that generally show good biocompatibility and is compatible with implantation in the future. Kidney proximal tubule cells and glomerular endothelial cells form polarized monolayers in our tubules, form their own extracellular matrix, and show functionality for important kidney transporters. In our preliminary results we showed initial work with induced pluripotent stem cell-derived kidney organoids, which is very promising for the development of implantable proximal tubule grafts. The aim was to develop vascularized kidney tubules for studying the clearance of protein bound uremic toxins, while gaining knowledge for the development of implantable kidney tubules. The developed models allow for studying transepithelial secretion of uremic toxins, an overarching application for these constructs. This will further advance our understanding of the kidney secretion processes and aid in studying interventions to main proximal tubule function in chronic kidney disease conditions in vitro. Furthermore, while fundamental by nature, our research deliverables enhance mechanistic insight in additive manufacturing and kidney development processes. Altogether, our findings will further advance the field of kidney engineering, while working towards kidney replacement therapies

Engineering the (vascularized) Proximal Tubule, a Guide Towards Functionality

This thesis focused on the fabrication of kidney proximal tubule models. A coaxial printing system allowed the robust and straightforward fabrication of coiled perfusable microfibers, replicating the kidney proximal convoluted tubules. The formed alginate-gelatin microfibers offer a complex tubular shape in which the cells exhibit mature markers, such as functional transporters and polarized monolayers. Moreover, this model has proven to support both healthy and cystinotic proximal tubule cell lines and allows for mechanistically studying tubulopathies. Though distinctive 3D-printing strategies offer opportunities to mimic the structure of the convoluted proximal tubule, printing resolution remains restricted for soft hydrogel materials. Therefore, a unique method was studied, whereby post-printing treatment induced resolution enhancement of methacrylated hyaluronic acid / gelatin methacryloyl (HAMA/GelMA) based hydrogels. This shrinking technique opens a wide application to create higher resolutions for 3D printing. However, we also found that the polycationic shrinking reagents used were relatively toxic for our cells of interest. Therefore, more research is needed to prevent the direct exposure of cells to the shrinking reagents. Using melt electrowriting, scaffolds were fabricated that are self-supportive, yet small-sized and highly porous. These scaffolds enable direct access to the basolateral and luminal cell sides to facilitate solute exchange with vasculature in immediate proximity, which is critical for functional proximal tubule constructs. The polymer applied, polycaprolactone, is used to prepare biomaterials that generally show good biocompatibility and is compatible with implantation in the future. Kidney proximal tubule cells and glomerular endothelial cells form polarized monolayers in our tubules, form their own extracellular matrix, and show functionality for important kidney transporters. In our preliminary results we showed initial work with induced pluripotent stem cell-derived kidney organoids, which is very promising for the development of implantable proximal tubule grafts. The aim was to develop vascularized kidney tubules for studying the clearance of protein bound uremic toxins, while gaining knowledge for the development of implantable kidney tubules. The developed models allow for studying transepithelial secretion of uremic toxins, an overarching application for these constructs. This will further advance our understanding of the kidney secretion processes and aid in studying interventions to main proximal tubule function in chronic kidney disease conditions in vitro. Furthermore, while fundamental by nature, our research deliverables enhance mechanistic insight in additive manufacturing and kidney development processes. Altogether, our findings will further advance the field of kidney engineering, while working towards kidney replacement therapies

Renal Tubular- and Vascular Basement Membranes and their Mimicry in Engineering Vascularized Kidney Tubules

The high prevalence of chronic kidney disease leads to an increased need for renal replacement therapies. While there are simply not enough donor organs available for transplantation, there is a need to seek other therapeutic avenues as current dialysis modalities are insufficient. The field of regenerative medicine and whole organ engineering is emerging, and researchers are looking for innovative ways to create (part of) a functional new organ. To biofabricate a kidney or its functional units, it is necessary to understand and learn from physiology to be able to mimic the specific tissue properties. Herein is provided an overview of the knowledge on tubular and vascular basement membranes' biochemical components and biophysical properties, and the major differences between the two basement membranes are highlighted. Furthermore, an overview of current trends in membrane technology for developing renal replacement therapies and to stimulate kidney regeneration is provided

Renal Tubular- and Vascular Basement Membranes and their Mimicry in Engineering Vascularized Kidney Tubules

The high prevalence of chronic kidney disease leads to an increased need for renal replacement therapies. While there are simply not enough donor organs available for transplantation, there is a need to seek other therapeutic avenues as current dialysis modalities are insufficient. The field of regenerative medicine and whole organ engineering is emerging, and researchers are looking for innovative ways to create (part of) a functional new organ. To biofabricate a kidney or its functional units, it is necessary to understand and learn from physiology to be able to mimic the specific tissue properties. Herein is provided an overview of the knowledge on tubular and vascular basement membranes' biochemical components and biophysical properties, and the major differences between the two basement membranes are highlighted. Furthermore, an overview of current trends in membrane technology for developing renal replacement therapies and to stimulate kidney regeneration is provided

Sacrificial Bioprinting of A Mammary Ductal Carcinoma Model

Cancer tissue engineering has remained challenging due to the limitation of the conventional biofabrication techniques to model the complex cancer microenvironment. Here we report the utilization of a sacrificial bioprinting strategy to generate biomimetic mammary duct-like structures within a hydrogel matrix, which was further populated with breast cancer cells, to model the genesis of ductal carcinoma and its subsequent outward invasion. This bioprinted mammary ductal carcinoma model provides a proof-of-concept demonstration of the value of using the sacrificial bioprinting technique for engineering biologically relevant cancer models, which may be possibly extended to other cancer types where duct-like structures are involved. This article is protected by copyright. All rights reserved

Sacrificial Bioprinting of A Mammary Ductal Carcinoma Model

Cancer tissue engineering has remained challenging due to the limitation of the conventional biofabrication techniques to model the complex cancer microenvironment. Here we report the utilization of a sacrificial bioprinting strategy to generate biomimetic mammary duct-like structures within a hydrogel matrix, which was further populated with breast cancer cells, to model the genesis of ductal carcinoma and its subsequent outward invasion. This bioprinted mammary ductal carcinoma model provides a proof-of-concept demonstration of the value of using the sacrificial bioprinting technique for engineering biologically relevant cancer models, which may be possibly extended to other cancer types where duct-like structures are involved. This article is protected by copyright. All rights reserved

Topographic Guidance in Melt-Electrowritten Tubular Scaffolds Enhances Engineered Kidney Tubule Performance

Contains fulltext : 230618.pdf (publisher's version ) (Open Access

Development and validation of bioengineered intestinal tubules for translational research aimed at safety and efficacy testing of drugs and nutrients

Currently used intestinal cell models have limited translational value, therefore, development of novel in vitro intestinal models that recapitulate the human in vivo setting more closely are of interest. Here, an advanced intestinal model was developed by the incorporation of physiological parameters, such as extracellular matrix (ECM) elements and shear stress, to cultured Caco-2 cells in a 3-dimensional environment. Caco-2 cells grown on ECM-coated hollow fiber membranes (HFM) under physiological shear stress show an improved phenotype, as demonstrated by the presence of enterocytes, goblet, Paneth, enteroendocrine and stem cells. Additionally, this model showed signs of an improved morphology due to the appearance of villi-like structures. Similar to epithelial cells grown on Transwells™, the current model remains easy to use, cost efficient and allows apical and basolateral access. The bioengineered intestinal tubule was validated by exposure to Clostridium difficile toxin A, the leading cause of healthcare-associated diarrhea. The loss of the tight junction network was supported by an increase in inulin-FITC leakage and the number of goblet cells increased, in agreement with clinical findings. In addition to toxicity screening, the bioengineered intestinal tubules are considered useful for drug and nutrient safety and efficacy testing