TGI PVG, adipose-derived stem cell-coated vascular graft for peripheral vascular disease

Microvessel endothelial cells are isolated from canine falciform ligament fat following an upper midline laparotomy. The fat is minced with scissors and incubated at 37°C for 30 min with collagenase (8 mg/mL) and human serum albumin (8 mg/mL) in Dulbecco’s divalent cation free phosphate buffered saline [DCF-PBS]) in a 50 ml Erlenmeyer flask. The resultant slurry of cells is then centrifuged, resulting in a vascular pellet and a buoyant layer of adipocytes. The vascular pellet is washed once by centrifugation with DCF-PBS containing bovine serum albumin (0.1% BSA). The final pellet is suspended in sodding medium (medium 199E without phenol red, containing autologous canine serum, 6:1). The average number of cells isolated per gram of fat is 1.08 X l0^6. Following resuspension of cells, 2 x l0^5 cells/cm2 are embedded within each sodded graft. The grafts are standard, clinically used grafts with a 4 mm internal diameter, thin walls, and external rings. Ten centimeter segments of 4 mm ePTFE grafts are capped at each end with Luer lock connectors and placed in a glass tube. Sodding medium is injected into the lumen of the graft 1 h prior to sodding. A total of approximately 10 mL sodding medium is injected into the lumen and through the interstices of the graft. The graft is then incubated for 1 h at room temperature. The sodding medium is then removed and replaced with the cell suspension. The graft is then pressurized to 5 psi. The total time necessary to perform cell isolation and graft sodding is approximately 1 h.