Method for perfusion decellularization of porcine whole liver and kidney for use as a scaffold for clinical-scale bioengineering engrafts
Method for perfusion decellularization of porcine whole liver and kidney for use as a scaffold for clinical-scale bioengineering engrafts
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DOI：
10.1111/xen.12141
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发表时间：
2015-01-01
影响因子：
3.9
通讯作者：
Bu, Hong
中科院分区：
文献类型：
--
作者：
Wang, Yujia;Bao, Ji;Bu, Hong
背景全器官工程为异种器官移植提供了一种新的供体来源。利用去污剂灌注法制备脱细胞的全器官支架是组织工程的一种策略。在这篇文章中，我们的目的是扩大脱细胞过程，以人类大小的肝脏和肾脏，以产生一个脱细胞基质与临床相关scale.MethodsWhole猪肝脏和肾脏脱细胞灌注使用不同的洗涤剂（1%SDS、1%Triton X-100、1%过氧乙酸（PAA）和1%NaDOC）分别经肝和肾的门静脉和肾动脉。在用PBS冲洗以去除去污剂后，对获得的肝和肾细胞外基质（ECM）进行组织学处理、残余细胞含量分析和ECM组分评价，以研究脱细胞效率、异种抗原去除和ECM保存。和细胞外基质成分的保存。相比之下，在使用Triton X-100、PAA和NaDOC产生的其他脱细胞组中观察到细胞碎片。猪肝、肾ECM的特殊染色和免疫组化进一步证实了Triton X-100和NaDOC组ECM的三维超微结构被破坏。此外，Triton X-100有效地消除了SDS处理组中残留的SDS，这确保了支架对细胞没有细胞毒性。因此，我们已经开发出一种最佳的方法，可以扩大使用与其他固体整体organs.ConclusionsOur SDS灌注协议可用于猪肝脏和肾脏去细胞化，以获得器官支架清除细胞材料，异种免疫原，并保存重要的ECM成分。
BackgroundWhole-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.MethodsWhole 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.ResultsThe 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.ConclusionsOur 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.