Jennifer Huling

Enhanced re-endothelialization of acellular kidney scaffolds for whole organ engineering via antibody conjugation of vasculatures

Decellularization of whole organs, such as the kidney hold great promise in addressing donor shortage for transplantation. However, successful implantation of engineered whole kidney constructs has been challenged by the inability to maintain endothelial cell coverage of the vasculature matrix, resulting in excessive blood clots, loss of vascular patency, and cell death within the construct. In this study, we describe an endothelial cell seeding approach that permits effective coating of the vascular matrix of the decellularized porcine kidney scaffold using a combination of static and ramping perfusion cell seeding. Furthermore, conjugation of CD31 antibodies to the vascular matrix improved endothelial cell retention on the vasculatures, which enhanced vascular patency of the implanted scaffold. These results demonstrate that our endothelial cell seeding method combined with antibody conjugation improves endothelial cell attachment and retention leading to vascular patency of tissue-engineered whole kidney in vivo.

Fabrication of biomimetic vascular scaffolds for 3D tissue constructs using vascular corrosion casts

UNLABELLED Vascularization is among the most pressing technical challenges facing tissue engineering of 3D organs. While small engineered constructs can rely solely on vascular infiltration and diffusion from host tissues following implantation, larger avascular constructs do not survive long enough for vessel ingrowth to occur. To address this challenge, strategies for pre-vascularization of engineered constructs have been developed. Various biofabrication techniques have been utilized for pre-vascularization, but limitations exist with respect to the size and complexity of the resulting vessels. To this end, we developed a simple and novel fabrication method to create biomimetic microvascular scaffolds using vascular corrosion casting as a template for pre-vascularization of engineered tissue constructs. Gross and electron microscopic analysis demonstrates that polycaprolactone (PCL)-derived kidney vascular corrosion casts are able to capture the architecture of normal renal tissue and can serve as a sacrificial template for the creation of a collagen-based vascular scaffold. Histological analysis demonstrates that the collagen vascular scaffolds are biomimetic in structure and can be perfused, endothelialized, and embedded in hydrogel tissue constructs. Our scaffold creation method is simple, cost effective, and provides a biomimetic, tissue-specific option for pre-vascularization that is broadly applicable in tissue engineering. STATEMENT OF SIGNIFICANCE Tissues in the body are vascularized to provide nutrients to the cells within the tissues and carry away waste, but creating tissue engineered constructs with functional vascular networks has been challenging. Current biofabrication techniques can incorporate blood vessel-like structures with straight or simple branching patterns into tissue constructs. Unfortunately, these techniques are expensive, complicated and create simplified versions of the complex vessel structures seen in native tissue. Our technique uses novel vascular corrosion casts of normal tissue as templates to create vascular scaffolds that are a copy of normal vessels. These vascular scaffolds can be easily incorporated into 3D tissue constructs. Our process is simple, inexpensive and inherently tissue-specific, making it widely applicable in the field of tissue engineering.

Comparing adult renal stem cell identification, characterization and applications

Despite growing interest and effort, a consensus has yet to be reached in regards to the identification of adult renal stem cells. Organ complexity and low turnover of renal cells has made stem cell identification difficult and lead to the investigation of multiple possible populations. In this review, we summarize the work that has been done toward finding and characterizing an adult renal stem cell population. In addition to giving a general overview of what has been done, we aim to highlight the variation in methods and outcomes. The methods used to locate potential stem cell populations can vary widely, but even within the relatively standard practice of BrdU labeling of slowly dividing cells, there are significant differences in protocols and results. Additional diversity exists in cell marker profiles and apparent differentiation potential seen in potential stem cell sources. Cataloging the variety of methods and outcomes seen so far may help to streamline future investigation and stear the field toward consensus. But even without firmly defined populations, the application of renal stem cells holds tantalizing potential. Populations of highly proliferative, multipotent cells of renal origin show the ability to engraft in injured kidneys, mitigate functional loss and occasionally show the ability to generate nephrons de novo. The progress toward regenerative medicine applications is also summarized.

Comparative analysis of two porcine kidney decellularization methods for maintenance of functional vascular architectures

Kidney transplantation is currently the only definitive solution for the treatment of end-stage renal disease (ESRD), however transplantation is severely limited by the shortage of available donor kidneys. Recent progress in whole organ engineering based on decellularization/recellularization techniques has enabled pre-clinical in vivo studies using small animal models; however, these in vivo studies have been limited to short-term assessments. We previously developed a decellularization system that effectively removes cellular components from porcine kidneys. While functional re-endothelialization on the porcine whole kidney scaffold was able to improve vascular patency, as compared to the kidney scaffold only, the duration of patency lasted only a few hours. In this study, we hypothesized that significant damage in the microvasculatures within the kidney scaffold resulted in the cessation of blood flow, and that thorough investigation is necessary to accurately evaluate the vascular integrity of the kidney scaffolds. Two decellularization protocols [sodium dodecyl sulfate (SDS) with DNase (SDS + DNase) or Triton X-100 with SDS (TRX + SDS)] were used to evaluate and optimize the levels of vascular integrity within the kidney scaffold. Results from vascular analysis studies using vascular corrosion casting and angiograms demonstrated that the TRX + SDS method was able to better maintain intact and functional microvascular architectures such as glomeruli within the acellular matrices than that by the SDS + DNase treatment. Importantly, in vitro blood perfusion of the re-endothelialized kidney construct revealed improved vascular function of the scaffold by TRX + SDS treatment compared with the SDS + DNase. Our results suggest that the optimized TRX + SDS decellularization method preserves kidney-specific microvasculatures and may contribute to long-term vascular patency following implantation. STATEMENT OF SIGNIFICANCE Kidney transplantation is the only curative therapy for patients with end-stage renal disease (ESRD). However, in the United States, the supply of donor kidneys meets less than one-fifth of the demand; and those patients that receive a donor kidney need life-long immunosuppressive therapy to avoid organ rejection. In the last two decades, regenerative medicine and tissue engineering have emerged as an attractive alternative to overcome these limitations. In 2013, Song et al. published the first experimental orthotopic transplantation of a bioengineering kidney in rodents. In this study, they demonstrated evidences of kidney tissue regeneration and partial function restoration. Despite these initial promising results, there are still many challenges to achieve long-term blood perfusion without graft thrombosis. In this paper, we demonstrated that perfusion of detergents through the renal artery of porcine kidneys damages the glomeruli microarchitecture as well as peritubular capillaries. Modifying dynamic parameters such as flow rate, detergent concentration, and decellularization time, we were able to establish an optimized decellularization protocol with no evidences of disruption of glomeruli microarchitecture. As a proof of concept, we recellularized the kidney scaffolds with endothelial cells and in vitro perfused whole porcine blood successfully for 24 h with no evidences of thrombosis.

MP74-06 FABRICATION OF BIOMIMETIC VASCULAR SCAFFOLDS USING VASCULAR CORROSION CASTS FOR RECONSTRUCTION OF KIDNEY TISSUES

inside the thermal container (TAUCt). The temperatures were measured every 30 minutes for 48 hours. RESULTS: Each of the technical variations were tested five times. There were no differences between groups regarding TAUCp and Tideal (p 1⁄4 0.550 and p1⁄40,053, respectively). The median Tideal in all groups was 0.5h (0-47.5h). Group 3 had longer Tnadir than groups 5 and 6 (p 1⁄4 0.018). The group 5 presented temperature mean area under the curve inside the thermal container (TAUCt) higher than groups 1, 2 and 6 (p 1⁄4 0.008). CONCLUSIONS: Cold storage, regardless of tested technique, results in temperatures outside the ideal range most of the time. The use of a metal case may delay the time to reach ideal temperature. Although the use of bars of ice causes higher temperatures within the thermal container, this may represent a greater approximation to the ideal temperature range.

Chapter 42 – Decellularized Whole Organ Scaffolds for the Regeneration of Kidneys

Decellularized extracellular matrix has long been applied as a scaffold material in tissue engineering. Recent investigation into decellularized whole solid organs has shown that the remaining extracellular matrix preserves the underlying architecture and protein organization inherent to the tissue. Decellularized whole organ scaffolds are of particular interest to renal regeneration owing to the complex nature of the kidneys because the extracellular matrix contains intact vascular and tubular networks. This chapter summarizes recent developments in whole organ scaffolds for kidney regeneration.