Date of Award
Open Access Thesis
Medical Doctor (MD)
Christopher K. Breuer
Edward L. Snyder
Medicine, Biomedical engineering, Biology
Objective: Tissue engineered vascular grafts (TEVGs) are useful in the surgical treatment of congenital heart defects. The development of TEVGs requires seeding of scaffolds composed of biodegradable polymers with bone marrow-derived mononuclear cells (BM-MNCs). The most common method used to isolate BM-MNCs involves density centrifugation in Ficoll. This process requires an International Organization for Standardization (ISO) class 7 clean room, is labor intensive, time intensive, and susceptible to operator variability. A recently developed filtration-based method for BM-MNC isolation uses a closed, sterile, and disposable system that removes the need for a clean room, decreases processing time, and is operator-independent. This study compared the efficacy of each method of BM-MNC isolation by evaluating the viability of cells recovered using each method, and by assessing the biologic and structure equivalence between neo-vessels created from scaffolds seeded using cells isolated by either method.
Methods: BM-MNCs were isolated from the bone marrow of immunocompetent syngeneic C57BL/6 wild type mice by either density centrifugation in Ficoll or using a filter-based method. The cells were seeded onto scaffolds fabricated from a polyglycolic acid (PGA) mesh coated with a 50:50 copolymer sealant of poly-L-lactide-co-ε-caprolactone. Seeded scaffolds were incubated overnight and then implanted as inferior vena cava (IVC) interposition grafts in 10-week-old wild type mice (n = 23 for each group). Grafts were explanted at 2 weeks post-implantation for analysis.
Results: Significantly greater total (filter: 44.3 ± 12.6x106 cells/mouse versus density centrifugation: 24.8 ± 8.8x106 cells/mouse, p=0.02) and viable (filter: 32.8 ± 6.7x106 cells/mouse versus density centrifugation: 20.6 ± 8.7x106 cells/mouse, p=0.04) BM-MNCs were isolated using filtration versus density centrifugation-based isolation. There was no significant difference in graft patency (filter: 78% patency versus density centrifugation: 87% patency, p=0.7), luminal diameter (filter: 633 ± 131 μm versus density centrifugation: 620 ± 82.9 μm, p=0.72) or neointimal thickness (filter: 37.9 ± 11.2 μm versus density centrifugation: 37.9 ± 7.8 μm, p=0.99) between groups at explantation. There was also no significant difference in quantitative macrophage infiltration between the two methods at explantation (filter: 1887 ± 907.7 cells/mm2 versus density centrifugation: 2041 ± 1078 cells/mm2, p=0.59).
Conclusion: BM-MNCs isolated using density centrifugation or the filter-based method were biologically equivalent and TEVGs formed from scaffolds seeded by each method were structurally similar when examined up to 14-days post-implantation in our in vivo murine model.
Bagi, Paul S., "Isolation Of Bone Marrow Mononuclear Cells For Fabrication Of Tissue-Engineered Vascular Grafts: Evaluation Of Two Methods" (2015). Yale Medicine Thesis Digital Library. 1944.