Development of an apoptosis-assisted decellularization method for maximal preservation of nerve tissue structure

Abstract

Tissue decellularization has expanded the ability to generate non-immunogenic organ replacements for a broad range of health applications. Current technologies typically rely on the use of harsh agents for clearing cellular debris, altering the tissue structure and potentially diminishing the pro-regenerative effects. We have developed a method for effectively, yet gently, removing cellular components from peripheral nerve tissue while preserving the native tissue architecture. The novelty of this process is in the induction of programmed cell death - or apoptosis - via a general cytotoxin, thereby enabling antigen clearance using only hypertonic wash buffers. The resulting acellular nerve scaffolds are nearly identical to unprocessed tissue on a microscopic level and elicit low immune responses comparable to an isograft negative control in a model of subcutaneous implantation.

Tissue-Specific Decellularization Methods: Rationale and Strategies to Achieve Regenerative Compounds

“…Finally, chemical acid-base [33] and TBP [32] treatments are rarely used, because they are very aggressive toward the proteins of the matrix. Specifically, acid-base solutions damage collagen and TBP disrupts protein-protein bonds.…”

“…This treatment is commonly used in combination with an osmotic cell burst, which breaks the cells, facilitating cell waste removal by Triton X-200. Other detergents, such as SDS or Triton-100, are used without osmotic shock [33,171]. Other than these, nucleases have been used in combination with detergents and osmotic methods to ensure that the DNA is properly removed.…”

Development of novel apoptosis-assisted lung tissue decellularization methods

“…DNA fragmentation is a late-stage event during apoptosis, and can be detected by fluorescence staining of DNA fragments using a TUNEL (Terminal dUTP Nick End-Labeling) assay 19 . This assay was used on camptothecin-assisted decellularization of rat sciatic nerves by Cornelison et al 14 . The authors also reported that apoptosis-assisted decellularized nerves exhibited high cytocompatibility in vitro and immunocompatibility in vivo after subcutaneous implantation 14 .…”

“…This assay was used on camptothecin-assisted decellularization of rat sciatic nerves by Cornelison et al 14 . The authors also reported that apoptosis-assisted decellularized nerves exhibited high cytocompatibility in vitro and immunocompatibility in vivo after subcutaneous implantation 14 . Another marker of apoptosis is externalization of phosphatidylserine to facilitate phagocytosis of apoptotic cells 19 .…”

Development of novel apoptosis-assisted lung tissue decellularization methods

“…To remove these immunogenic cellular debris, harsh washing steps are required, which further creates a problem of compromising tissue integrity 12 . Recently, an alternate approach was suggested in which tissues are decellularized by means of programmed cell death, or apoptosis, in which cells fragment into apoptotic bodies containing intracellular debris; these apoptotic bodies allow easier removal of cellular debris [13][14][15] . This approach does not rely on heavy use of detergents and water washes to induce cell death, but instead generates apoptotic bodies that are easier to remove via gentle washes.…”