Mehran Abolbashari

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.

Repopulation of porcine kidney scaffold using porcine primary renal cells.

UNLABELLED The only definitive treatment for end stage renal disease is renal transplantation, however the current shortage of organ donors has resulted in a long list of patients awaiting transplant. Whole organ engineering based on decellularization/recellularization techniques has provided the possibility of creating engineered kidney constructs as an alternative to donor organ transplantation. Previous studies have demonstrated that small units of engineered kidney are able to maintain function in vivo. However, an engineered kidney with sufficient functional capacity to replace normal renal function has not yet been developed. One obstacle in the generation of such an organ is the development of effective cell seeding methods for robust colonization of engineered kidney scaffolds. We have developed cell culture methods that allow primary porcine renal cells to be efficiently expanded while maintaining normal renal phenotype. We have also established an effective cell seeding method for the repopulation of acellular porcine renal scaffolds. Histological and immunohistochemical analyses demonstrate that a majority of the expanded cells are proximal tubular cells, and the seeded cells formed tubule-like structures that express normal renal tubule phenotypic markers. Functional analysis revealed that cells within the kidney construct demonstrated normal renal functions such as re-adsorption of sodium and protein, hydrolase activity, and production of erythropoietin. These structural and functional outcomes suggest that engineered kidney scaffolds may offer an alternative to donor organ transplant. STATEMENT OF SIGNIFICANCE Kidney transplantation is the only definitive treatment for end stage renal disease, however the current shortage of organ donors has limited the treatment. Whole organ engineering based on decellularization/recellularization techniques has provided the possibility of creating engineered kidney constructs as an alternative to donor organ transplantation. While previous studies have shown that small units of engineered kidneys are able to maintain function in animal studies, engineering of kidneys with sufficient functional capacity to replace normal renal function is still challenging due to inefficient cell seeding methods. This study aims to establish an effective cell seeding method using pig kidney cells for the repopulation of acellular porcine kidney scaffolds, suggesting that engineered kidneys may offer an alternative to donor organ transplant.

Potential Use of Autologous Renal Cells from Diseased Kidneys for the Treatment of Renal Failure.

Chronic kidney disease (CKD) occurs when certain conditions cause the kidneys to gradually lose function. For patients with CKD, renal transplantation is the only treatment option that restores kidney function. In this study, we evaluated primary renal cells obtained from diseased kidneys to determine whether their normal phenotypic and functional characteristics are retained, and could be used for cell therapy. Primary renal cells isolated from both normal kidneys (NK) and diseased kidneys (CKD) showed similar phenotypic characteristics and growth kinetics. The expression levels of renal tubular cell markers, Aquaporin-1 and E-Cadherin, and podocyte-specific markers, WT-1 and Nephrin, were similar in both NK and CKD kidney derived cells. Using fluorescence- activated cell sorting (FACS), specific renal cell populations were identified and included proximal tubular cells (83.1% from NK and 80.3% from CKD kidneys); distal tubular cells (11.03% from NK and 10.9% from CKD kidneys); and podocytes (1.91% from NK and 1.78% from CKD kidneys). Ultra-structural analysis using scanning electron microscopy (SEM) revealed microvilli on the apical surface of cultured cells from NK and CKD samples. Moreover, transmission electron microscopy (TEM) analysis showed a similar organization of tight junctions, desmosomes, and other intracellular structures. The Na+ uptake characteristics of NK and CKD derived renal cells were also similar (24.4 mmol/L and 25 mmol/L, respectively) and no significant differences were observed in the protein uptake and transport characteristics of these two cell isolates. These results show that primary renal cells derived from diseased kidneys such as CKD have similar structural and functional characteristics to their counterparts from a normal healthy kidney (NK) when grown in vitro. This study suggests that cells derived from diseased kidney may be used as an autologous cell source for renal cell therapy, particularly in patients with CKD or end-stage renal disease (ESRD).

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.

Chapter 36 – Genitourinary System

The genitourinary system is constantly exposed to different adverse conditions that can lead to various degrees of structural and functional damage. Recent advances in regenerative medicine and tissue engineering have provided new therapeutic options for many patients. While several therapies have been successfully applied in the clinic, the majority of technologies are still in the development stage. It is anticipated that more of these developments will mature into therapies over time. This chapter gives an overview of current and new therapeutic developments in the genitourinary system.