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Decellularization and Recellularization Methodology for Human Saphenous Veins

Artikel i vetenskaplig tidskrift
Författare Vijay Kumar Kuna
Bo Xu
Suchitra Sumitran-Holgersson
Publicerad i Jove-Journal of Visualized Experiments
Nummer/häfte 137
ISSN 1940-087X
Publiceringsår 2018
Publicerad vid Institutionen för kliniska vetenskaper, Avdelningen för kirurgi
Språk en
Länkar dx.doi.org/10.3791/57803
Ämnesord Bioengineering, Tissue Engineering, Decellularization, Recellularization, Regenerative, endothelial progenitor cells, proof-of-concept, growth-factor, smooth-muscle, stem-cells, in-vivo, transplantation, proliferation, grafts, matrix
Ämneskategorier Transplantationskirurgi

Sammanfattning

Vascular conduits used during most vascular surgeries are allogeneic or synthetic grafts that often lead to complications caused by immunosuppression and poor patency. Tissue engineering offers a novel solution to generate personalized grafts with a natural extracellular matrix containing the recipients cells using the method of decellularization and recellularization. We show a detailed method for performing decellularization of the human saphenous vein and recellularization by perfusion of peripheral blood. The vein was decellularized by perfusing 1% Triton X-100, 1% tri-n-butyl-phosphate (TnBP) and 2,000 Kunitz units of deoxyribonuclease (DNase). Triton X-100 and TnBP were perfused at 35 mL/min for 4 h while DNase was perfused at 10 mL/min at 37 degrees C for 4 h. The vein was washed in ultrapure water and PBS and then sterilized in 0.1% peracetic acid. It was washed again in PBS and preconditioned in endothelial medium. The vein was connected to a bioreactor and perfused with endothelial medium containing 50 IU/mL heparin for 1 h. Recellularization was performed by filling the bioreactor with fresh blood, diluted 1:1 in Steen solution, and adding endocrine gland-derived vascular endothelial growth factors (80 ng/mL), basic fibroblast growth factors (4 mu L/mL), and acetyl salicylic acid (5 mu g/mL). The bioreactor was then moved into an incubator and perfused for 48 h at 2 mL/min while maintaining glucose between 3 - 9 mmol/L. Later, the vein was washed with PBS, filled with endothelial medium and perfused for 96 h in the incubator. Treatment with Triton X-100, TnBP and DNase decellularized the saphenous vein in 5 cycles. The decellularized vein looked white in contrast to normal and recellularized veins (light red). The hematoxylin & eosin (H&E) staining showed the presence of nuclei only in normal but not in decellularized veins. In the recellularized vein, H&E-staining showed the presence of cells on the lumina! surface of the vein.

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http://gu.se/forskning/publikation/?publicationId=271343
Utskriftsdatum: 2020-04-09