448. Therapeutic Angiogenesis by Subcutaneous Cell Sheet Delivery Is Superior to Cell Injection: A Study of ADSC Efficacy in a Model of Hind Limb Ischemia
Authors: Pavel I. Makarevich, Maria A. Boldyreva, Anastasia Yu. Efimenko, Evgeny V. Gluhanyuk, Konstantin V. Dergilev, Julia O. Gallinger, Yu-Chen Hu, Yelena V. Parfyonova
Journal: Molecular Therapy (2016)

Engineering of cell sheets (CS) is an effective approach for delivery of cells to induce angiogenesis and tissue regeneration. Basis for increased CS efficacy is better engraftment and cell survival due to absence of anoikis and intact cell-to-cell interaction in the transplant. Still question to be addressed is whether CS are superior to injection route in terms of efficacy/cell engraftment and how can we improve therapeutic output of CS delivery for stimulation of tissue repair. We conducted a comparative study of adipose-derived stromal cells (ADSC) delivery in a model of hind-limb ischemia. C57/B6 male mice with unilateral limb ischemia (n=8-10/group) were injected with 106 of passage 3 syngeneic ADSC or transplanted with equivalent amount of cells in CS shape. After that animals were monitored for limb perfusion by laser-Doppler for 2 wks and then euthanized for histology studies of vessel density and ADSC detection using a PKH26 or CMFDA fluorescent label. Obtained samples were stained for macrophage invasion, endothelial cell markers and proliferation/apoptosis to evaluate cell fate. Our animal test data has revealed that by Day 14 delivery of ADSC by means of injection induced restoration of limb perfusion compared to negative control group (41.5±4.7% vs. 29.7±3.0% respectively; p=0.01) indicating well-known pro-angiogenic properties of these cells. Still, subcutaneous transplantation of CS was found to be superior to injection in terms of perfusion. CS-treated animals had the highest (55.3±7.3; p=0.03 vs. injected ADSC) perfusion by the end of experiment. This data was supported by vascular density assessment, which revealed increased capillary counts in both ADSC-treated groups with significantly higher values after CS delivery compared to injection (220.9±11.4 vs. 191.3±8.8 respectively; p=0.01). Analysis of necrotic tissue span in hematoxylin/eosin-stained section found a significant decrease of necrosis in ADSC-treated animals and also found CS to have better performance in terms of tissue protection compared to injection. Furthermore, we also evaluated ADSC engraftment and found that after injection pre-labeled cells reside as scattered mass and found their number to decrease over time by Day 14. Whilst after CS transplantation the cells were compactly localized in the site of application. CS were found to be vascularized by capillary vessels and infiltrated by CD68+ macrophage indicating graft-host interaction. Interestingly, certain cells within CS were found to show signs of proliferation (Ki67+) with sporadic apoptosis (cleaved caspase-3+) with overall transplant staying intact and viable by Day 14 after delivery. Overall, our data indicates that transplantation of CS is superior to injection of equivalent amount of cells. We may speculate that this is not limited to ADSC and can be utilized for novel treatment methods. In an attempt to enhance the CS efficacy we turned our attention to hybrid constructs consisting of ADSC and endothelial cells to generate pre-vascularized constructs. Our preliminary data revealed "tube-like" behavior of HUVEC seeded on top of a CS from ADSC and may be the way to overcome the diffusion distance issue and generate CS with vascularized structure, which have a closer resemblance to native tissue structures. Moreover, this unveils the possibility to generate multi-layered constructs and imitate cell-to-cell interaction for basic and applied studies.