Perfusion decellularization of porcine esophagus: Study of two processing factors affecting the folded mucosal structure of the esophageal scaffold

Abstract

Acellular scaffolds from decellularized donor organs are showing promising clinical results in tissue and organ repair and regeneration. A successful decellularization process is determined by (a) its capability to decellularize complete organs of large animals, (b) retention of the extracellular matrix (ECM) structures and morphologies, and (c) minimal loss of ECM proteins. In this study, porcine esophagi were perfused in full thickness with 0.25% w/v sodium dodecyl sulfate at perfusion rates 0.1–0.2 ml/min for up to 5 days. Decellularized tissues were characterized for their residual DNA, histological staining for their matrix structures, immunohistochemical staining for collagen type IV and laminin, and scanning electron microscopy for structural integrity. Our results showed that full thickness esophageal tissues treated using the horizontal perfusion setup were decellularized with good structural and biochemical integrity in the ECM. Residual DNA content in decellularized tissues was found to be 36 ± 12 ng/mg of tissues (n = 6) which was significantly lower than that of native tissues (p = .00022). Our study showed that the organ must be decellularized in full thickness and perfusion pressure must be controlled to minimize radial expansion. These factors were found to be critical in preserving the folded mucosa in the decellularized tissues.

Decellularization of porcine esophageal tissue at three diameters and the bioscaffold modification with EETs‐ECM gel

“…Tissue swelling after alkaline treatment and detergent perfusion are adequate to meet with two of the commonly accepted metrics of decellularization, that is, the lack of cell nuclei stained with the H&E protocol and the residual amount of DNA. Effective tissue decellularization and prevention of unfolding of the complaint mucosa are consistent with the perfusion method applied to the esophagus 7 . Meanwhile, the modification of the DSC traces after tissue decellularization is consistent with the necessary balance between the selected methods/agents with the characteristics of the resulting matrix; the presence of the denaturation peak suggests preservation of collagen stability.…”

“…A second perfusion was applied to samples (sodium deoxycholate 4% in PBS, 2×, RT) during 1.5 h for 1 day esophagus or 2.5 h for 21 or 45 h esophagus, followed by a washing (PBS, 4°C, overnight). The perfusions were carried out in horizontal position 7 Third step: nucleases treatment .…”

“…Decellularization of tissues from different sources using different procedures makes a difference to the composition, properties, and performance of a biologic scaffold. Decellularization of the esophagus is commonly carried out by perfusion of different surfactants, which benefits from the simplicity and good control of the process 1,2,7–9 . Among other requirements, the bioscaffold type, the extent of surgery and the species used for tissue decellularization has also been associated with significant heterogeneity in pathological outcomes after implantation 10 .…”

Quantitative Phase Imaging to Study the Effect of Sodium Dodecyl Surfactant on Adherent L929 Fibroblasts on Tissue Culture Plates

“…Sodium dodecyl sulfate (SDS) is one of the most effective and commonly used surfactants to decellularize tissues and organs. Our previous study confirmed that SDS effectively decellularized porcine esophagi [6]. However, the real-time interactions between SDS and cells during decellularization are not well understood, except that the process is very rapid.…”

“…The SDS solution was prepared in PBS (Sigma-Aldrich, Saint Louis, MO, USA) at a concentration of 200 ppm (w/v). The SDS concentrations used in organ decellularization typically ranged between 1000 to 100,000 ppm [6,15,16]. However, at these SDS concentrations, their effects on the cells were too rapid to be imaged using our current QPI setup.…”