The Effect of a Novel Whole Liver Decellularization Protocol on the Extracellular Matrix and Cell Components Residuals
1 Department of Hepatobiliary Surgery, The First Affiliated Hospital of Wenzhou Medical College, Wenzhou 325000, China
2 Laboratory of Internal Medicine, The First Affiliated Hospital of Wenzhou Medical College, Wenzhou 325000, China
Abstract：There has been a conflict between preservation of extracellular matrix and reduction of donor’s cell components on decellularized scaffold production in tissue engineering. Therefore a protocol for decellularization is the key to solve this problem. We developed a novel decellularization protocol which used TritonX-100 as a major agent for decellularizing assisted by SDS, enzymes, freezethaw processing, hypotonic and hypertonic solutions etc. After morphological and histological observation through immunohistochemistry and scanning electron microscope, evaluation of ECM content by ELISA, using absorbance detection and Westernblotting to test cell components residuals, and immunofluorescence after 3D perfusion culture of WBF-344 cells in the scaffold, the results showed that, by using this new protocol, three dimensional structure like collagen, laminin, fibronectin appeared well protected, and the extracellular matrix contents of decellularized scaffold such as GAG, HGF and VEGF was 90% and 40% of normal tissue. Besides a DNA elimination, MCHI and HMGB-1 were only 10% of normal tissue. WBF-344 cells grew well in the scaffold and the albumin level was rising. In conclusion, this study built a novel decellularization protocol and evaluation system, and proposed a promising application of the acellular liver scaffold in stem cell differentiation and liver disease therapy.
