Cell therapy for transplantations in cardiovascular system: decellularization and recellularization of porcine pericardium

摘要

Introduction: Immune systems response, which leads to degeneration of allo/xenogeneic aortic heart valves, is the biggest problem of organ-transplantations generally. The immune response is caused by the presence of donor cells in the host organism. There is an effort to find the way, how to diminish this complication. The aim of our study was to prepare a graft made of decellularized porcine pericardium and to recolonize it with adipose tissue-derived stem cells. This graft will be later mechanically stimulated in a bioreactor. Materials and methods: For the decellularization of porcine pericardium we used varied combinations, concentrations, and exposure time of sodium deoxycholate (SDC), sodium dodecyl sulfate (SDS) and Triton X 100. The degree of decellularization process was assessed by hematoxylin-eosin staining and Hoechst staining of histological samples. Biocompatibility of the tissue extracts was evaluated using xCelligence method with rat vascular smooth muscle cells. The decellularized pericardium was seeded with adipose tissue-derived stem cells and cultured for 11 days. Results and discussion: Using various combinations, concentrations and time of incubation on native porcine pericardium we prepared pericardium with a various degree of decellularization. Because of the extremely toxicity of SDS after decellularization, the pericardium was washed out thoroughly to remove the rest of the chemicals used. The Cell-Viability Assay showed very low toxicity of decellularizated pericardium after the washing. For the recellularization we chose pericardium without residual cells. Adipose tissue-derived stem cells seeded on the pericardium attached, spread, and proliferated during an eleven-day culture. These grafts will be further mechanically stimulated in a bioreactor and both stem cell differentiation and their production of extracellular matrix will be assessed. Fig. 1. (A) A histological section of native porcine pericardium stained with Hoechst 33342 (blue - cell nuclei). (B) A histological section of decellularized porcine pericardium stained with Hoechst 33342. Epifluorescence microscope Olympus IX51, DP70 camera, obj. ×10 (A, B). (C) Decellularized porcine pericardium seeded with adipose tissue-derived stem cells on day 5 after seeding, stained with Hoechst 33342 (blue - cell nuclei) and Phalloidin-TRIC (F-actin - red). Epifluorescence microscope Olympus IX71, DP71 camera, obj. ×10(C). Conclusion: We prepared the decellularized porcine pericardium, which allowed adipose tissue-derived stem cells attachment, spreading and proliferation. This method could be important for development of grafts for transplantations in cardiovascular system.