Plastic Surgery Research Council
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PSRC 60th Annual Meeting
Program and Abstracts

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Elimination of Reperfusion-induced Microcirculatory Alterations in-vivo by ASC Supernatant without ASC Cell
Wei Z. Wang, M.D., Xin-Hua Fang, M.T., Shelley J. Williams, B.S., Linda L. Stephenson, M.T., Richard C. Baynosa, M.D., Kayvan T. Khiabani, M.D., William A. Zamboni, M.D..
University of Nevada School of Medicine, Las Vegas, NV, USA.

Purpose: Our pilot studies demonstrated that human adipose-derived stem cells (ASCs) cultured with expansion medium produced a significant amount of vascular endothelial growth factor (VEGF) in the supernatant and the levels of VEGF expression in ASC supernatants were in direct proportion to the number of ASC in the culture flask. In the present study, we hypothesized that ASCs might be able to secrete multiple cytokines in the supernatant in-vitro and ASC supernatant even without ASCs might have a significant impact in-vivo on ischemia/reperfusion(I/R)-induced microcirculatory alterations and endothelial dysfunction.
Methods: Fat tissues were surgically harvested from rat (n=6) bilateral flanks and processed for isolation of stromal vascular fraction (SVF). The adherent ASCs were harvested after 24h culture of SVF in non-hematopoietic expansion medium (NHEM). 1x106 ASCs were sub-cultured with 5ml of NHEM in T-25 culture flask for 3, 6, 9 and 12 days without passaging but with medium change every 3 days. The post-cultivated medium was collected from flask every 3 days. After centrifugation, the supernatant was collected and stored in -20ºC. Eight oxidative stress cytokines including TNFα, TGFβ, MCP-1, IL-1α, IL-1β, IL-6, IL-15 and VEGF were simultaneously and quantitatively measured in ASC supernatants that collected at day 9. The effect of ASC supernatant on I/R-induced microcirculatory alterations was examined in the vascular pedicle isolated rat cremaster model underwent 4 hours of ischemia followed by 2 hour of reperfusion. Immediately after reperfusion, ASC supernatant (n=6) collected at day 9 or NHEM (n=6, control) was infused into systemic circulation at speed 1ml/min/kg for 10mins through femoral vein cannulation.
Results: ELISA study showed that ASC supernatant contains multiple highly expressed cytokines (Fig-1). The average concentration of TNFα, TGFβ, IL-1α, IL-6, and VEGF was significantly higher than the control (P < 0.05). The average concentration of IL-6, in particular, was 5-fold higher as compared to the control (P = 0.0006). I/R-induced vasospasm, arteriole stagnation and capillary no-reflow that often appear in early phase of reperfusion were eliminated by the administration of ASC supernatant (Fig-2).
Conclusions: 1. ASCs in cell culture produced significant amount of cytokines in the supernatant including IL-6 and VEGF. 2. ASC supernatant even without ASC could be used as a paracrine agent to interfere with I/R-induced microcirculatory alterations and endothelial dysfunction.


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