Perfusion decellularization and characterization of esophagus with preserved folded mucosa for clinical reconstruction

Esophageal diseases necessitate the use of replacements or scaffolds to repair the esophagus. As porcine esophagus is structurally and biochemically similar to that of human’s, decellularized porcine scaffold provides the potential for esophageal regeneration. This thesis focuses on the development of perfusion processes to decellularize porcine esophagi. The first part of the thesis evaluates on the decellularization process. Our objectives include producing scaffolds with preserved folded mucosa and basement membrane, retention of the structural extracellular matrices (ECM) and ECM proteins. Two important processing criteria found in our study were (1) full thickness esophagus must be used, and (2) the perfusion pressure must be controlled to minimize radial expansion. The vertical and the horizontal perfusion setups were compared. Vertically perfused scaffolds resulted in the unfolding of the mucosa, but for the horizontal process, the folded mucosa was preserved. Full decellularization of the native esophagus was achieved with 0.25% w/v sodium dodecyl sulfate (SDS) at perfusion rates 0.1-0.2 ml/min for up to 5 days. Both native and decellularized scaffolds were characterized and compared. Characterisations include histological examinations, optical and electron microscopy, and residual DNA analysis. The second part of this thesis focused on the scaffold cytotoxicity evaluated according to ISO 10993 Part 5, and in vitro cell-scaffold interaction. Our results showed that the metabolic activity of L929 fibroblasts cultured for 1,3, and 7 days in extracted culture medium and standard medium (negative control) were not statistically significant (i.e. P > .05). Cell-scaffolds interaction results showed that the scaffold supported proliferation of mammalian cells. Further study showed that the scaffold also supported TR 146 epithelial proliferation and expression of the keratin for up to 28 days. The final section of this thesis evaluates the use of quantitative phase imaging (QPI) technique for qualitative and quantitative imaging of cell-surfactant interactions in real-time. Interactions between phosphate buffered saline, deionized water, and SDS solution on adherent L929 fibroblasts on culture plates were imaged and analysed. QPI results showed that decellularization using SDS involved the rapid rupture of the cell membrane followed by the detachment of cellular debris from the substrate.Doctor of Philosoph

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

Decellularization is the process of removing cellular components from native tissues or organs to obtain an acellular, collagenous scaffold for use in tissue engineering and organ regeneration. Surfactants are widely used to produce acellular scaffolds for clinical applications. However, cell–surfactants interactions have not been studied in depth. Cell-surfactant interaction was studied in a time-lapsed manner using sodium dodecyl sulfate (SDS) solution (surfactant) on adherent L929 fibroblasts as a test solution, phosphate-buffered saline (PBS) solution as control solution (isotonic), and deionized water as positive test solution (hypotonic), respectively. The QPI results show changes in the relative height and cross-sectional area of the cells, with various test solutions and exposure times. In particular, it was observed that the removal of the cell with SDS involved the disruption of the cellular membrane and detachment of the cell contents from the adhering surface. This study demonstrated the feasibility of using the QPI technique to understand the decellularization process