Cryopreservation Allows for Extended Graft Survival in a Rat Orthotopic Hind Limb Model
1Department of Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine, Baltimore, MD, 2Department Of Comparative Medicine, Medical University of South Carolina, Charleston, SC, 3Tissue Testing Technologies LLC, Charleston, SC
Meeting: 2019 American Transplant Congress
Abstract number: B28
Keywords: Graft survival, Preservation, Rat
Session Information
Session Name: Poster Session B: Ischemia Reperfusion & Organ Rehabilition
Session Type: Poster Session
Date: Sunday, June 2, 2019
Session Time: 6:00pm-7:00pm
Presentation Time: 6:00pm-7:00pm
Location: Hall C & D
*Purpose: Vascularized composite allotransplantation (VCA) is a viable reconstructive option for devastating tissue defects. 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 better donor matching and provide additional time to pre-condition recipients for novel immunomodulatory regimens. We present first outcomes for cryopreservation in a rat hind limb model.
*Methods: Lewis rats served as donors (7) and recipients (14) of orthotopic hind limb transplants. Limbs were flushed with heparinized Lactated Ringers, loaded with 10% DMSO in Culture Medium, cooled at 1oC/min to -90oC and stored below -130oC for 1-2 weeks. Non-frozen controls (N=2) were performed. Intervention groups: controlled cooling rate frozen limbs with A) Spontaneous nucleation (N=8), B) Induced nucleation at -4oC (N=4). Thawed and tissue DMSO concentration reduced limbs were transplanted at the mid-thigh. Recipients were monitored daily until the study endpoints of Post-Operatieve Day (POD) 14 and 30. Biopsies were acquired at POD7 and endpoint. H&E stained samples were histologically reviewed. Pre-implanted limbs were evaluated for tissue viability using a metabolic resazurin assay.
*Results: Post-rewarming femoral arteries, skin and cartilage were >70% of fresh control viability; muscle was 35-40% (p<0.05). All control limb transplants were viable at POD14. All transplanted limbs established blood flow. 1 of 8 spontaneous nucleation limbs was viable at POD7. Histopathology revealed regeneration of skeletal myofibers and associated fibrosis at POD10. 2 of 4 induced nucleation limbs (50%) demonstrated gross signs of healing around the ankles and feet by POD7 and further skin and muscle regeneration between POD7-30.
*Conclusions: We report the first above-knee cryopreserved rat limb graft survival after frozen storage. Further improvement in muscle viability is needed. Between POD7-30 limbs grossly improved demonstrating that the cryopreserved tissues had regenerative capacity. These studies constitute critical first steps toward VCA banking.
To cite this abstract in AMA style:
Fidder SA, Oh B, Messner F, Furtmueller G, Helke K, Greene E, Chen Z, Campbell L, Brandacher G, Brockbank K. Cryopreservation Allows for Extended Graft Survival in a Rat Orthotopic Hind Limb Model [abstract]. Am J Transplant. 2019; 19 (suppl 3). https://atcmeetingabstracts.com/abstract/cryopreservation-allows-for-extended-graft-survival-in-a-rat-orthotopic-hind-limb-model/. Accessed May 16, 2025.« Back to 2019 American Transplant Congress