Abstract 227: Adipose Stromal Vascular Fraction (SVF) Vasculogenesis Demonstrates a Dose Response Through the Assembly of Endothelial Networks

Abstract

Objective Critical limb ischemia (CLI) is a challenging clinical entity complicated by chronic microvascular disease. Stromal vascular fraction (SVF), a heterogeneous population of cells derived from adipose tissue, has garnered interest as a means of an autologous cell-based approach to vasculogenesis in these patients. The SVF concentration needed to achieve this goal is still unknown. Our goal was to determine if SVF vasculogenesis exhibits a dose-response using in vitro and murine in vivo models. Methods SVF was isolated from adult male Sprague Dawley transgenic-GFP+ Rat epididymal fat pads and serially diluted using SVF growth media and then added to a respectively labeled matrigel-coated plates. Wells were then labeled with Griffonia simplicifolia-1 (GS-1) rhodamine lectin. Phase and fluorescent microscopy images of each well were then taken. The same SVF concentrations were suspended in collagen gels and subcutaneously implanted into the flanks of 6 Rag-1 deficient mice and imaged with confocal microscopy 2 weeks later to determine if a dose-response could be seen in-vivo. Tubule length was calculated using Metamorph software. Results Vasculogenic networks showed monolayering at concentrations of 5.0 x 10 5 and 1.0 x 10 6 cells/mL, where as the intermediate dose exhibited greater complexity to its network. Total tubule length, measured in μm, peaked with the 2.50 x 10 5 cells/mL concentration. Tubule length increased significantly up to this peak, and then gradually decreased at higher concentrations. GS-1 staining was positive when viewing the tubules under fluorescence microscopy. Preliminary results from the in-vivo model showed increasing vasculogenesis up to a maximal tubule length for the 1 x 10 6 cells/mL concentration. Conclusion SVF vasculogenesis exhibits a dose-response. Tubule formation is of endothelial phenotype and tubule length is directly proportional to cell concentration up to a peak concentration of 2.5 x 10 5 cells/mL. Preliminary in-vivo data also suggests that a dose-reponse exists. We are encouraged by these results that suggest that cell based therapy may be tailored for appropriate dosing in CLI patients.