Decellularization and Recellularization Methodology for Human Saphenous Veins

Abstract

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 recipient's 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 °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 µL/mL), and acetyl salicylic acid (5 µg/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 luminal surface of the vein.

Tissue-Engineered Grafts from Human Decellularized Extracellular Matrices: A Systematic Review and Future Perspectives

“…The researchers also considered the decellularization of veins, including umbilical cord vein, [181,183,193,194,195,196,197,198,199,200,201,202,203,204], saphenous vein [205,206], femoral vein [207], veins of the hand dorsum [15], and of amputated legs [208].…”

“…Finally, Mangold et al [197] exposed the samples to (1) Triton X-100/SDS/IGEPAL-CA630, (2) NaCl and dH 2 O, and (3) peroxyacetic acid in ethanol, prior to performing the DNase-I/RNase treatment; however, none of the above methods were adequate to support endothelial cell attachment and growth without conditioning. Recently, both the saphenous and the femoral veins were decellularized by perfusion with a method based on Triton X-100, tri-n-butyl-phosphate (TnBP), and DNase [206,207]; while Porzionato et al [15], as previously described for arteries of the hand dorsum, also treated the veins with dH 2 O, Trypsin/EDTA and Triton X-100/ammonium hydroxide with a final dH 2 O soaking phase.…”

“…Many methods were adopted to achieve scaffolds colonization and many kind of cells were also seeded on/into flat [194,195,197] or tubular scaffolds [193,196,197,198,199,201,202,204,206,207] in static [194,195,197,198,199,201] or dynamic conditions (i.e., rotational or mechanical stimulation) [199,201,204] including perfusion [193,196,202,206,207]. It is noteworthy that some authors also considered the functional suitability of the developed supports.…”

“…According to the literature, the cell populations considered included primary human fibroblasts and vascular smooth muscle cells [193], human gingival fibroblasts [194], and vascular endothelial cells [195]; umbilical vein endothelial cells [195,197,202], also in mixture with human cord blood-derived endothelial progenitor cells (EPC) [204], MSCs [198,199,201], and adipose-derived stem cells differentiated into endothelial-like cells, were transfected with eNOS [209]. Recellularization was also attempted by whole blood [196,206,207] with different outcomes. In fact, some authors demonstrated that the strategy is convenient and easy, as its use also avoids long cell expansion times, eventual spontaneous mutations, and discomfort for the patient [196,206]; conversely, Rambol et al [207] did not gain good results as the method failed in veins re-endothelization.…”

“…Recellularization was also attempted by whole blood [196,206,207] with different outcomes. In fact, some authors demonstrated that the strategy is convenient and easy, as its use also avoids long cell expansion times, eventual spontaneous mutations, and discomfort for the patient [196,206]; conversely, Rambol et al [207] did not gain good results as the method failed in veins re-endothelization.…”