Development of an apoptosis-assisted decellularization method for maximal preservation of nerve tissue structure.
Development of an apoptosis-assisted decellularization method for maximal preservation of nerve tissue structure.
复制标题
开发细胞凋亡辅助脱细胞方法，最大限度地保存神经组织结构。
DOI：
10.1016/j.actbio.2018.07.009
复制
发表时间：
2018-09-01
影响因子：
9.7
通讯作者：
Schmidt CE
中科院分区：
文献类型：
--
作者：
Cornelison RC;Wellman SM;Park JH;Porvasnik SL;Song YH;Wachs RA;Schmidt CE
在优化组织和器官脱细胞方法时，组织结构的保存往往是首要目标。然而，许多现行的方案依赖于洗涤剂，这些洗涤剂有助于提取细胞成分，但也会破坏组织结构。利用一种内在的细胞过程，如细胞凋亡，并在不使用有害试剂的情况下促进细胞去除，可能更有益。在细胞凋亡过程中，细胞从细胞外基质脱离，降解其内部成分，并将其内容物碎片化以便更容易清除。我们开发了一种方法，利用这一过程仅使用温和的洗涤缓冲液实现组织脱细胞。我们已经证明，用喜树碱处理外周神经组织可诱导细胞凋亡的早期标志物——裂解的半胱天冬酶 - 3表达，以及晚期标志物——TUNEL + DNA片段化。然后，在高渗磷酸盐缓冲盐水中温和洗涤，接着进行DNA酶处理，以依赖细胞凋亡的方式清除细胞成分。这种洗涤模式没有显著影响胶原蛋白或糖胺聚糖含量，但根据条件培养基实验，它足以去除任何细胞毒性化合物的痕迹。所得的无细胞组织移植物在体内具有免疫耐受性，并呈现出完整的基膜微观结构，类似于天然的、未经处理的神经。因此，体外诱导细胞凋亡是一种有前途的组织脱细胞方法，它不使用刺激性试剂，同时更好地保留了天然组织的优势，如组织特异性成分和微观结构。
Preservation of tissue structure is often a primary goal when optimizing tissue and organ decellularization methods. Many current protocols nonetheless rely on detergents that aid extraction of cellular components but also damage tissue architecture. It may be more beneficial to leverage an innate cellular process such as apoptosis and promote cell removal without the use of damaging reagents. During apoptosis, a cell detaches from the extracellular matrix, degrades its internal components, and fragments its contents for easier clearance. We have developed a method that leverages this process to achieve tissue decellularization using only mild wash buffers. We have demonstrated that treating peripheral nerve tissue with camptothecin induced both an early marker of apoptosis, cleaved caspase-3 expression, as well as a late stage marker, TUNEL+ DNA fragmentation. Clearance of the cellular components was then achieved in an apoptosis-dependent manner using a gentle wash in hypertonic phosphate buffered saline followed by DNase treatment. This wash paradigm did not significantly affect collagen or glycosaminoglycan content, but it was sufficient to remove any trace of the cytotoxic compound based on conditioned media experiments. The resulting acellular tissue graft was immunogenically tolerated in vivo and exhibited an intact basal lamina microarchitecture mimicking that of native, unprocessed nerve. Hence, ex vivo induction of apoptosis is a promising method to decellularize tissue without the use of harsh reagents while better preserving the benefits of native tissue such as tissue-specific composition and microarchitecture.
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DOI：
10.1073/pnas.1411975111
发表时间：
2014-12-09
影响因子：
11.1
作者：
Bourgine, Paul E.;Scotti, Celeste;Martin, Ivan
通讯作者：
Martin, Ivan
影响因子：
3
作者：
Gillies, Allison R.;Smith, Lucas R.;Varghese, Shyni
通讯作者：
Varghese, Shyni
影响因子：
--
作者：
Hudson, TW;Liu, SY;Schmidt, CE
通讯作者：
Schmidt, CE
影响因子：
14
作者：
Bourgine, Paul E.;Pippenger, Benjamin E.;Martin, Ivan
通讯作者：
Martin, Ivan
影响因子：
19
作者：
Bourgine, Paul Emile;Gaudiello, Emanuele;Martin, Ivan
通讯作者：
Martin, Ivan