Multiphoton Microscopy for Detection of GFP labeled Mesenchymal Stem Cells in Porcine AAA Model

Abstract

IntroductionMultiphoton microscopy has transcended from a picture‐taking novelty to a powerful tool used to detect excitation fluorescence. This abstract aims to assess the ability to detect green fluorescence protein (GFP) labeled porcine mesenchymal stem cells (MSC) that were infused into aneurismal aortic tissue.Methods/ResultsMSCs were isolated from Yorkshire swine bone marrow and infected with GFP + lentivirus. An aortic aneurysm was created in the pig (by mechanical and chemical degradation) and GFP labeled MSC were introduced into the aneurismal aorta. 24 hours after implantation, the pig was sacrificed and the aorta was removed. The tissue was secured in agar and sent for multiphoton imaging. Imaging was performed at 0, 24, 48, 72, and 96 hrs after harvest. Multiphoton imaging detected GFP labeled cells with high fluorescence excitability in the aortic tissue up to 72hrs after tissue harvest. With second harmonic generation, collagen molecules were imaged and were distinctly different from the GFP excited cells. Presence of GFP positive cells was confirmed with subsequent immunohistochemistry and western blot.ConclusionsMultiphoton microscopy is useful in tracking GFP labeled MSCs infused into the aneurismal aorta of swine. These cells can be tracked up to 72hrs after tissue harvest with presence of GFP by western blot. This powerful tool can be applied to in vitro tracking of in vivo placed MSCs.Grant Funding Source: National Institute of Health K08HL073313