Extended Graft Survival After Cryopreservation and Storage Below -130°C in a Rat Orthotopic Hind Limb Model
Samuel A.J. Fidder, M.D.1,2, Byoungchol Oh, D.V.M., Ph.D1, Franka Messner, M.D.1, Georg J. Furtmueller, M.D.1, Kristi Helke, D.V.M., Ph.D3, Elizabeth D. Greene4, Zhen Chen, Msc.4, Lia H. Campbell, Ph.D.4, Gerald Brandacher, M.D.1, Kelvin G.M. Brockbank, Ph.D.4,5.
1Johns Hopkins Hospital, Baltimore, MD, USA, 2Erasmus University Medical Center, Rotterdam, Netherlands, 3Medical University of South Carolina, Charleston, SC, USA, 4Tissue Testing Technologies LLC, North Charleston, SC, USA, 5Clemson University, Charleston, SC, USA.
Purpose: Vascularized composite allotransplantation (VCA) is an increasingly used reconstructive option for devastating tissue defects of the face, hand, arm, and most recently the penis. Though highly successful cases have been reported, the growth of the field is restrained by the limited ischemia time that is tolerated by the graft. Long-term graft preservation would allow for transplantation across greater distances and thus improved organ sharing, better donor matching, and provide transplant teams with additional time to pre-condition recipients for novel immunomodulatory regimens. Here we present our first outcomes using cryopreservation in a rat hind limb transplant model. Methods: Lewis rats were used as both donors (7) and recipients (14) of orthotopic hind limb transplants. Limbs were flushed with Lactated Ringers containing Heparin, loaded with cryoprotectant formulation (10% DMSO in Culture Medium), cooled at 1oC/min to -90oC and then stored below -130oC for 1-2 weeks. Controls (N=2) were performed without cryopreservation. Intervention groups were A) controlled cooling rate frozen limbs with spontaneous nucleation (N=8) and B) similarly treated limbs with induced nucleation at -4oC (N=4). Transplants were thawed and tissue DMSO concentrations reduced by simultaneously soaking and perfusing the limbs with culture medium containing 0.5M mannitol. Preserved syngeneic hind limbs were then transplanted at the mid-thigh to the recipient animal using a non-suture cuff technique for vascular anastomosis. Recipients were monitored daily until the study endpoint of POD14. Biopsies were acquired at postoperative day (POD) 7 and endpoint. Samples were stained with hematoxylin and eosin for histopathology review. Control and treated limbs were also evaluated without implantation to determine tissue component viability using a metabolic resazurin assay. Results: Viability evaluation after rewarming demonstrated that the femoral arteries, skin and cartilage were >70% of fresh controls, while the muscle was 35-40% of controls (p<0.05). Blood flow was established in all transplanted limbs. Both control limb transplants were successful to POD14. Only one of eight limbs cryopreserved using spontaneous nucleation survived past POD7 and the recipient was euthanized on POD10. Histopathology revealed regeneration of skeletal myofibers and associated fibrosis. Two of four limbs cryopreserved using induced nucleation (50%) demonstrated gross signs of healing around the ankles and feet by POD7 (Figure 1) and further skin and muscle regeneration between POD7-POD14. Conclusions: This is the first demonstration of above knee cryopreserved rat limb graft survival after frozen storage. Further improvement in muscle cryopreservation is needed. From POD7 onwards the limbs grossly improved demonstrating that the cryopreserved limb tissues had the capacity to regenerate. These studies constitute critical first steps toward banking of complex VCAs to enable better phenotypic and immunologic matching of donor tissues to recipients.
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