Residual Detergent Detection Method for Nondestructive Cytocompatibility Evaluation of Decellularized Whole Lung Scaffolds

Abstract

The development of reliable tissue engineering methods using decellularized cadaveric or donor lungs could potentially provide a new source of lung tissue. The vast majority of current lung decellularization protocols are detergent based and incompletely removed residual detergents may have a deleterious impact on subsequent scaffold recellularization. Detergent removal and quality control measures that rigorously and reliably confirm removal, ideally utilizing nondestructive methods, are thus critical for generating optimal acellular scaffolds suitable for potential clinical translation. Using a modified and optimized version of a methylene blue-based detergent assay, we developed a straightforward, noninvasive method for easily and reliably detecting two of the most commonly utilized anionic detergents, sodium deoxycholate (SDC) and sodium dodecyl sulfate (SDS), in lung decellularization effluents. In parallel studies, we sought to determine the threshold of detergent concentration that was cytotoxic using four different representative human cell types utilized in the study of lung recellularization: human bronchial epithelial cells, human pulmonary vascular endothelial cells (CBF12), human lung fibroblasts, and human mesenchymal stem cells. Notably, different cells have varying thresholds for either SDC or SDS-based detergent-induced cytotoxicity. These studies demonstrate the importance of reliably removing residual detergents and argue that multiple cell lines should be tested in cytocompatibilitybased assessments of acellular scaffolds. The detergent detection assay presented here is a useful nondestructive tool for assessing detergent removal in potential decellularization schemes or for use as a potential endpoint in future clinical schemes, generating acellular lungs using anionic detergent-based decellularization protocols.

Characterization of decellularized ovine small intestine submucosal layer as extracellular matrix‐based scaffold for tissue engineering

“…Moreover, SEM results showed that cells were attached and expanded efficiently after being in culture for 24 h. These results represent that high viability and less toxicity of decellularized scaffolds depend on various factors such as the percentage of applied detergent and an efficient washing process. These results are in accordance to those reported by Sullivan et al On the contrary, Zvarova et al have shown that an excessive washing step does not affect the viability and toxicity. In addition, Wang et al have shown that TX‐100 can expel the remained SDS of the tissue and reduces the toxicity; however, the results of this study are contrary to that reported by Wang.…”

Crossing kingdoms: Using decellularized plants as perfusable tissue engineering scaffolds

“…The decellularization solutions used in this study contain high concentrations of detergents that were chosen after an initial investigation that accounted for the time needed for decellularization. Residual detergents present after decellularization have been shown to affect cellular viability [41] and thus could become problematic with use of high detergent concentrations. There were at best only limited viability issues in this study, nevertheless, this and other potential concerns should be addressed with further refinement of the decellularization techniques.…”

Comparative biology of decellularized lung matrix: Implications of species mismatch in regenerative medicine

“…To date, studies by our group and others have demonstrated feasibility of decellularized human, primate, pig and rat tissues in achieving some of these features (5, 18, 24, 29, 60-63). In this work, we demonstrated that the basement membrane (required to separate the blood supply), and the gross tissue architecture needed to provide gas exchange were maintained in each of our decellularized species.…”