Development of an Apoptosis-Assisted Decellularization Method for Maximal Preservation of Nerve Tissue Structure: Apoptosis-Assisted Decellularization for Maximal Nerve Tissue Preservation

摘要

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.