An efficient method for decellularization of the rat liver.

BACKGROUND/PURPOSE: Using gradient ionic detergent, we optimized the preparation procedure for the decellularized liver biologic scaffold, and analyzed its immunogenicity and biocompatibility. METHODS: EDTA, hypotonic alkaline solution, Triton X-100, and gradient sodium dodecyl sulfate (1%, 0.5%, and 0.1%, respectively) were prepared for continuous perfusion through the hepatic vascular system. The decellularization of the liver tissue was performed with the optimized reagent buffer and washing protocol. In addition, the preservation of the original extracellular matrix was observed. To analyze its biocompatibility, the scaffold was embedded in a heterologous animal and the inflammation features, including the surrounding cell infiltration and changes of the scaffold architecture, were detected. The cell-attachment ability was also validated by the perfusion culture of HepG2 cells with the scaffold. RESULTS: By using gradient ionic detergent, we completed the decellularization process in approximately 5 h, which was shorter than >10 hours in previous experiments (p<0.001). The extracellular matrix was kept relatively intact, with no obvious inflammatory cellular infiltration or structural damage in the grafted tissue. The engraftment efficiencies of HepG2 were 86±5% (n=8). The levels of albumin and urea synthesis were significantly superior to the ones in traditional two-dimensional culture. CONCLUSION: The current new method can be used efficiently for the decellularization of the liver biologic scaffold with satisfying biocomparability for application both in vivo and in vitro.

In vitro culture of tumour-derived hepatocytes in decellularised whole-liver biological scaffolds.

BACKGROUND: To explore the feasibility of preparing transplantable recellularised liver grafts by preparing a decellularised whole-liver scaffold, decellularisation and recellularisation of a liver scaffold using a chemical detergent were performed on the liver cancer cell lines HepG2 and C3A. MATERIALS AND METHODS: D-Hanks' solution containing sodium EDTA, gradient sodium dodecyl sulphate (SDS, 1/0.5/0.1%) and Triton X-100 were infused via the portal vein to prepare the decellularised scaffold. HepG2 and C3A cells were seeded onto the scaffold, and a circulation perfusion culture was carried out. The efficiency of the engraftment and the function of the cells in the scaffold were evaluated. RESULTS: The decellularisation method used completely removed the intrahepatic cellular components while only components with low immunogenicity were retained, such as collagen and fibronectin. Meanwhile, the vascular structure was completely retained, which provided a structural basis for circulation bypass. The engraftment efficiencies of the HepG2 and C3A cells were 86 ± 5 and 88 ± 5%, respectively, and were not significantly different between the two groups (p > 0.05, n = 10). The cells grew well on the scaffold, and the albumin synthesis and urea secretion functions were superior to those obtained after a traditional two-dimensional culture. CONCLUSIONS: The preparation of a liver scaffold using a chemical detergent technique has good reproducibility, and the scaffold is suitable for the growth of liver tumour cell lines.

[Preparation of a decellularized rat liver scaffold and its biocompatibility].

OBJECTIVE: To develop a novel method for preparing decellularized liver biological scaffold (DLBS) for liver tissue engineering. METHODS: DLBS was prepared by treatment of rat livers with detergent and enzymatic cell extraction and observed under optical and scanning electron microscopes. To assess the biocompatibility of the product, C3A cells and bone marrow-derived mesenchymal cells (BM-MSCs) were cocultured with DLBS as the scaffold, and the effect of DLBS on the proliferation of C3A cells was evaluated by MTT assay. DLBS was also implanted under the dorsal skin of SD rats to evaluate the tissue biocompatibility of this material. RESULTS: Application of the detergent and enzymatic extraction allowed full extraction of the cells in the liver, leaving an extracellular matrix scaffold composed mainly of collagen and elastic fibrin. The coculture experiment showed that C3A cells and BM-MSCs could grow on and adhere to DLBS. The result of MTT assay showed that DLBS could promote the proliferation of C3A cells. CONCLUSION: This cell-free DLBS, which retains intact extracellular matrix and promotes cell attachment, proliferation, growth and differentiation, can be an ideal biological matrix scaffold material.