Analysis of decellularized mouse liver fragment and its recellularization with human endometrial mesenchymal cells as a candidate for clinical usage.

Abstract

Decellularized tissue has been used as a natural extracellular matrix (ECM) or bioactive biomaterial for tissue engineering. The present study aims to compare and analyze different decellularization protocols for mouse liver fragments and cell seeding and attachment in the created scaffold using human endometrial mesenchymal cells (hEMCs).After collecting and dissecting the mouse liver into small fragments, they were decellularized by Triton X-100 and six concentrations of sodium dodecyl sulfate (SDS; 0.025, 0.05, 0.1, 0.25, 0.5, and 1%) at different exposure times. The morphology and DNA content of decellularized tissues were studied, and the group with better morphology and lower DNA content was selected for additional assessments. Masson's tri-chrome and periodic acid Schiff staining were performed to evaluate ECM materials. Raman confocal spectroscopy analysis was used to quantify the amount of collagen type I, III and IV, glycosaminoglycans and elastin. Scanning electron microscopy and MTT assay were applied to assess the ultrastructure and porosity and cytotoxicity of decellularized scaffolds, respectively. In the final step, hEMCs were seeded on the decellularized scaffold and cultured for one week, and finally the cell attachment and homing were studied morphologically.The treated group with 0.1% SDS for 24 h showed a well preserved ECM morphology similar to native control and showing the minimum level of DNA. Raman spectroscopy results demonstrated that the amount of collagen type I and IV was not significantly changed in this group compared to the control, but a significant reduction in collagen III and elastin protein levels was seen (P < 0.001). The micrographs showed a porous ECM in decellularized sample similar to the native control with the range of 2.25 µm to 7.86 µm. After cell seeding, the infiltration and migration of cells in different areas of the scaffold were seen. In conclusion, this combined protocol for mouse liver decellularization is effective and its recellularization with hEMCs could be suitable for clinical applications in the future.