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Optimizing perfusion-decellularization methods of porcine livers for clinical-scale whole-organ bioengineering.

ABSTRACT: Aim. To refine the decellularization protocol of whole porcine liver, which holds great promise for liver tissue engineering. Methods. Three decellularization methods for porcine livers (1% sodium dodecyl sulfate (SDS), 1% Triton X-100 + 1% sodium dodecyl sulfate, and 1% sodium deoxycholate + 1% sodium dodecyl sulfate) were studied. The obtained liver scaffolds were processed for histology, residual cellular content analysis, and extracellular matrix (ECM) components evaluation to investigate decellularization efficiency and ECM preservation. Rat primary hepatocytes were seeded into three kinds of scaffold to detect the biocompatibility. Results. The whole liver decellularization was successfully achieved following all three kinds of treatment. SDS combined with Triton had a high efficacy of cellular removal and caused minimal disruption of essential ECM components; it was also the most biocompatible procedure for primary hepatocytes. Conclusion. We have refined a novel, standardized, time-efficient, and reproducible protocol for the decellularization of whole liver which can be further adapted to liver tissue engineering.

SUBMITTER: Wu Q

PROVIDER: S-EPMC4396818 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Optimizing perfusion-decellularization methods of porcine livers for clinical-scale whole-organ bioengineering.

Wu Qiong Q Bao Ji J Zhou Yong-jie YJ Wang Yu-jia YJ Du Zheng-gui ZG Shi Yu-jun YJ Li Li L Bu Hong H

BioMed research international 20150331

Aim. To refine the decellularization protocol of whole porcine liver, which holds great promise for liver tissue engineering. Methods. Three decellularization methods for porcine livers (1% sodium dodecyl sulfate (SDS), 1% Triton X-100 + 1% sodium dodecyl sulfate, and 1% sodium deoxycholate + 1% sodium dodecyl sulfate) were studied. The obtained liver scaffolds were processed for histology, residual cellular content analysis, and extracellular matrix (ECM) components evaluation to investigate de ...[more]

PMID: 25918720

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Project description:Background:Ex vivo machine perfusion (MP) can better preserve organs for transplantation. We have recently reported on the first application of an MP protocol in which liver allografts were fully oxygenated, under dual pressures and subnormothermic conditions, with a new hemoglobin-based oxygen carrier (HBOC) solution specifically developed for ex vivo utilization. In those studies, MP improved organ function post-operatively and reduced inflammation in porcine livers. Herein, we sought to refine our knowledge regarding the impact of MP by defining dynamic networks of inflammation in both tissue and perfusate. Methods: Porcine liver allografts were preserved either with MP (n = 6) or with cold static preservation (CSP; n = 6), then transplanted orthotopically after 9 h of preservation. Fourteen inflammatory mediators were measured in both tissue and perfusate during liver preservation at multiple time points, and analyzed using Dynamic Bayesian Network (DyBN) inference to define feedback interactions, as well as Dynamic Network Analysis (DyNA) to define the time-dependent development of inflammation networks. Results: Network analyses of tissue and perfusate suggested an NLRP3 inflammasome-regulated response in both treatment groups, driven by the pro-inflammatory cytokine interleukin (IL)-18 and the anti-inflammatory mediator IL-1 receptor antagonist (IL-1RA). Both DyBN and DyNA suggested a reduced role of IL-18 and increased role of IL-1RA with MP, along with increased liver damage with CSP. DyNA also suggested divergent progression of responses over the 9 h preservation time, with CSP leading to a stable pattern of IL-18-induced liver damage and MP leading to a resolution of the pro-inflammatory response. These results were consistent with prior clinical, biochemical, and histological findings after liver transplantation. Conclusion: Our results suggest that analysis of dynamic inflammation networks in the setting of liver preservation may identify novel diagnostic and therapeutic modalities.

Project description:Background & Aims:End-ischemic hypothermic oxygenated machine perfusion (HOPE) of the donor liver for 1-2 h mitigates ischemia-reperfusion injury during subsequent liver transplantation. Extended preservation time may be preferred to facilitate difficult recipient hepatectomy or to optimize logistics. We therefore investigated whether end-ischemic dual HOPE (DHOPE) could extend preservation time for up to 24 h using a porcine liver reperfusion model. Methods:Following 30 min warm ischemia, porcine livers were subjected to 2 h static cold storage (SCS), followed by 2 h, 6 h, or 24 h DHOPE (n = 6 per group). Subsequent normothermic reperfusion was performed for 4 h using autologous blood. Two livers preserved by 24 h SCS served as additional controls. A proof of principle confirmation was carried out in 2 discarded human livers subjected to extended DHOPE. Hepatocellular and cholangiocyte injury and function were assessed. Oxidative stress levels and histology were compared between groups. Results:Perfusion flows remained stable during DHOPE, regardless of duration. After normothermic reperfusion, livers perfused for 24 h by DHOPE had similar lactate clearance, blood pH, glucose, and alanine aminotransferase levels, and biliary pH, bicarbonate, and LDH levels, as livers perfused for 2 h and 6 h. Levels of malondialdehyde and high-mobility group box 1 in serum and liver parenchyma were similar for all groups. Histological analysis of bile ducts and liver parenchyma revealed no differences between the groups. Extended DHOPE in discarded human livers preserved hepatocellular and cholangiocyte function and histology after reperfusion. In contrast, livers preserved by 24 h SCS were non-functioning. Conclusion:Extended end-ischemic DHOPE enabled successful preservation of porcine and discarded human donor livers for up to 24 h. Extended DHOPE enables safe extension of preservation time, which may facilitate allocation and transplantation from a logistical perspective, and further expand the donor pool. Lay summary:It has been suggested that preserving liver grafts with a technique called (dual) hypothermic oxygenated machine perfusion ([D]HOPE) leads to better outcomes after transplantation than if livers are stored on ice, especially if an organ is of lesser quality. In this study, we showed that DHOPE could be used to preserve liver grafts for up to 24 h. This extended procedure could be used globally to facilitate transplantation and expand the donor pool.

Dataset Information

Optimizing perfusion-decellularization methods of porcine livers for clinical-scale whole-organ bioengineering.

Publications

Optimizing perfusion-decellularization methods of porcine livers for clinical-scale whole-organ bioengineering.

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