Decellularization methods of porcine kidneys for whole organ engineering using a high-throughput system.

End-stage renal failure is a devastating disease, with donor organ transplantation as the only functional restorative treatment. The current number of donor organs meets less than one-fifth of demand, so regenerative medicine approaches have been proposed as potential therapeutic alternatives. One such approach for whole large-organ bioengineering is to combine functional renal cells with a decellularized porcine kidney scaffold. The efficacy of cellular removal and biocompatibility of the preserved porcine matrices, as well as scaffold reproducibility, are critical to the success of this approach. We evaluated the effectiveness of 0.25 and 0.5% sodium dodecyl sulfate (SDS) and 1% Triton X-100 in the decellularization of adult porcine kidneys. To perform the decellularization, a high-throughput system was designed and constructed. In this study all three methods examined showed significant cellular removal, but 0.5% SDS was the most effective detergent (<50 ng DNA/mg dry tissue). Decellularized organs retained intact microarchitecture including the renal vasculature and essential extracellular matrix components. The SDS-treated decellularized scaffolds were non-cytotoxic to primary human renal cells. This method ensures clearance of porcine cellular material (which directly impacts immunoreactivity during transplantation) and preserves the extracellular matrix and cellular compatibility of these renal scaffolds. Thus, we have developed a rapid decellularization method that can be scaled up for use in other large organs, and this represents a step toward development of a transplantable organ using tissue engineering techniques.

Sullivan DC ，Mirmalek-Sani SH ，Deegan DB ，Baptista PM ，Aboushwareb T ，Atala A ，Yoo JJ ... -  《-》

Method for perfusion decellularization of porcine whole liver and kidney for use as a scaffold for clinical-scale bioengineering engrafts.

Whole-organ engineering provides a new alternative source of donor organs for xenotransplantation. Utilization of decellularized whole-organ scaffolds, which can be created by detergent perfusion, is a strategy for tissue engineering. In this article, our aim is to scale up the decellularization process to human-sized liver and kidney to generate a decellularized matrix with optimal and stable characteristics on a clinically relevant scale. Whole porcine liver and kidney were decellularized by perfusion using different detergents (1% SDS, 1% Triton X-100, 1% peracetic acid (PAA), and 1% NaDOC) via the portal vein and renal artery of the liver and kidney, respectively. After rinsing with PBS to remove the detergents, the obtained liver and kidney extracellular matrix (ECM) were processed for histology, residual cellular content analysis, and ECM components evaluation to investigate decellularization efficiency, xenoantigens removal, and ECM preservation. The resulting liver and kidney scaffolds in the SDS-treated group showed the most efficient clearance of cellular components and xenoantigens, including DNA and protein, and preservation of the extracellular matrix composition. In comparison, cell debris was observed in the other decellularized groups that were generated using Triton X-100, PAA, and NaDOC. Special staining and immunochemistry of the porcine liver and kidney ECMs further confirmed the disrupted three-dimension ultrastructure of the ECM in the Triton X-100 and NaDOC groups. Additionally, Triton X-100 effectively eliminated the residual SDS in the SDS-treated group, which ensured the scaffolds were not cytotoxic to cells. Thus, we have developed an optimal method that can be scaled up for use with other solid whole organs. Our SDS-perfusion protocol can be used for porcine liver and kidney decellularization to obtain organ scaffolds cleared of cellular material, xenoimmunogens, and preserved vital ECM components.

Wang Y ，Bao J ，Wu Q ，Zhou Y ，Li Y ，Wu X ，Shi Y ，Li L ，Bu H ... -  《-》

Porcine kidneys as a source of ECM scaffold for kidney regeneration.

To produce and examine decellularized kidney scaffolds from porcine as a platform for kidney regeneration research. Porcine kidneys were decellularized with sodium dodecyl sulfate solution and Triton X-100 after the blood was rinsed. Then the renal ECM scaffolds were examined for vascular imaging, histology to investigate the vascular patency, degree of decellularization. Renal ECM scaffolds of porcine kidneys were successfully produced. Decellularized renal scaffolds retained intact microarchitecture including the renal vasculature and essential extracellular matrix components. We have developed an excellent decellularization method that can be used in large organs. These scaffolds maintain their basic components, and show intact vasculature system. This represents a step toward development of a transplantable organ using tissue engineering techniques.

Guan Y ，Liu S ，Liu Y ，Sun C ，Cheng G ，Luan Y ，Li K ，Wang J ，Xie X ，Zhao S ... -  《-》

Biocompatibility and hemocompatibility of efficiently decellularized whole porcine kidney for tissue engineering.

Whole kidney decellularization is a promising approach in regenerative medicine for engineering a functional organ. The reaction of the potential host depends on the biocompatibility of these decellularized constructs. Despite the proven ability of decellularized kidney scaffolds to guide cell attachment and growth, little is known about biocompatibility and hemocompatibility of these scaffolds. Our aim is to prepare decellularized kidneys of a clinically relevant size and evaluate its biocompatibility and hemocompatibility. Porcine kidneys were cannulated via the renal artery, and then perfused with 0.1% sodium dodecyl sulfate solution. Hematoxylin and eosin as well as DAPI staining confirmed cellular clearance from native kidneys in addition to preservation of the microstructure. SEM confirmed the absence of any cellular content within the scaffold, which is maintained in a well-organized 3D architecture. Decellularized kidneys retained the intact renal vasculature upon examination with contrast radiography. The essential structural extracellular matrix molecules were well-preserved. Scaffolds were susceptible to enzymatic degradation upon collagenase treatment. Scaffolds showed a good hemocompatibility when exposed to porcine blood. Decellularization was efficient to remove 97.7% of DNA from native kidneys in addition to the immunogenic and pathogenic antigens. Scaffolds did not induce the human immune response in vitro. Decellularized kidneys were non-cytotoxic to pig kidney cells (PKs). PKs were able to grow and proliferate within the decellularized renal scaffolds with maintaining a higher function than cells grown as monolayers. Thus, we have developed a rapid decellularization technique for generating biocompatible kidney scaffolds that represents a step toward development of a transplantable organ. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2034-2047, 2018.

Hussein KH ，Saleh T ，Ahmed E ，Kwak HH ，Park KM ，Yang SR ，Kang BJ ，Choi KY ，Kang KS ，Woo HM ... -  《-》

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. 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.

Zambon JP ，Ko IK ，Abolbashari M ，Huling J ，Clouse C ，Kim TH ，Smith C ，Atala A ，Yoo JJ ... -  《-》