Comparison of aortic valve allograft decellularization techniques in the rat
Comparison of aortic valve allograft decellularization techniques in the rat
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DOI：
10.1002/jbm.a.30777
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发表时间：
2006-11-01
影响因子：
4.9
通讯作者：
Ross, David B.
中科院分区：
文献类型：
--
作者：
Meyer, Steven R.;Chiu, Brian;Ross, David B.
啮齿动物模型对于理解同种异体主动脉瓣移植（AVAs）免疫介导的衰竭是必不可少的。脱细胞被认为可以降低AVAs的免疫原性。本研究的目的是确定用于大鼠模型的AVAs脱细胞的最有效方法。在Lewis主动脉瓣中比较了三种不同的脱细胞技术。洗涤剂脱细胞包括一系列低渗和高渗Tris缓冲液，每缓冲液含0.5% Triton X-100 48小时，然后在磷酸盐缓冲盐水中冲洗72小时。除不添加Triton X-100外，渗透脱细胞的方法与基于洗涤剂的技术类似。酶解细胞包括胰蛋白酶/EDTA在37℃下48小时。通过光镜（H&E, Movat's五色体）、免疫组织化学检测残余细胞元素和羟脯氨酸检测进行评估。基于洗涤剂的方法除了保存细胞外基质（ECM）外，还几乎完全去除了小叶和主动脉壁的细胞。渗透裂解与ECM的保存和适度的脱细胞有关。酶解脱细胞导致完全脱细胞，但广泛的变性和ECM碎裂。当植入同种异体大鼠的肾下主动脉1周后，用洗涤剂和渗透法去除细胞的瓣膜未能刺激同种异体免疫反应，因为没有T细胞浸润。低剂量洗涤剂的渗透裂解方案似乎在去除抗原细胞元素和保存ECM方面最有效。(c) 2006 Wiley期刊公司
Rodent models have been essential to understanding the immune-mediated failure of aortic valve allografts (AVAs). Decellularization has been proposed to reduce the immunogenicity of AVAs. The objective of this study was to determine the most effective method to decellularize AVAs for use in a rat model. Three different decellularization techniques were compared in Lewis aortic valves. Detergent decellularization involved a series of hypotonic and hypertonic Tris buffers at VC for 48 h/buffer containing 0.5% Triton X-100 followed by a 72 h washout in phosphate-buffered saline. Osmotic decellularization was performed in similar manner to the detergent-based technique except without the addition of Triton X-100. Enzymatic decellularization consisted of trypsin/EDTA at 37 degrees C for 48 h. Assessment was performed with light microscopy (H&E, Movat's pentachrome), immunohistochemistry for residual cellular elements, and hydroxyproline assays. Detergent-based methodology effected near-complete decellularization of both the leaflets and aortic wall in addition to preservation of the extracellular matrix (ECM). Osmotic lysis was associated with preservation of ECM and moderate decellularization. Enzymatic decellularization resulted in complete decellularization but extensive degeneration and fragmentation of the ECM. When implanted into the infrarenal aorta of allogeneic rats for 1 week, valves decellularized with detergent-based and osmotic methodology failed to stimulate an allogeneic immune response as evidenced by an absence of T cell infiltrates. Osmotic lysis protocols with low dose detergent appear to be most effective at both removing antigenic cellular elements and preserving ECM. (c) 2006 Wiley Periodicals, Inc.