Abstract: Directional Freezing and Vitrification of Whole Limbs for Future Transplantation and Organ Banking

Abstract

INTRODUCTION: According to the World Health Organisation, less than 10% of humanities needs for transplantable organs are being met. No data is available for vascularized composite tissue allotransplantations (VCA), yet these cases are further complicated by the need for an instantly available and compatible recipient. VCA containing skin, fat, blood vessels, bone, bone marrow and nerve are the ultimate tool available to date in reconstructive surgery. Widespread use of this tool is hindered in part by the availability of limbs which can survive a limited amount of time disconnected from the body. Cryobiology is the scientific field which investigates biology at low temperatures. Cryobiologists have long sought to cryopreserve biological samples ranging from single cells to complex organs and whole animals. Vitrification is the transformation of a substance into a non-crystalline amorphous solid using rapid heat removal. Directional freezing is a technique, consisting of highly controlled and cell-friendly ice crystal morphology that significantly reduces mechanical damage. Extremely efficient heat removal and controlled ice crystal propagation make it suitable for freezing both small and large volumes alike. Combining Directional freezing and VCA may open the door for “Organ Banking”. If composite tissue could be frozen and then thawed without damage, non transplanted tissue and organ waste would be reduced, potentially enabling better donor and recipient availability and match. METHODS: We used directional freezing and vitrification on a syngeneic heterotrophic rat hindlimb transplant model and monitored the animals and transplanted limbs for up to 72 hours. Cell and tissue samples were taken for culture and histology. RESULTS: Immediately upon thawing the donor hindlimb blood vessels and tissue felt similar to the recipient’s. Following revascularization reperfusion was clinically evident by color and bleeding from distal sites to the anastomosis. Limb survival was noted up to 72 hours post op. Tissue samples including muscle, skin and blood vessels where taken at 24, 48 and 72 hours from transplantation. Histology demonstrated viable myocytes, intact intimal lining of blood vessels and all skin layers. CONCLUSION: Whole limb freezing and reimplantation using directional freezing and vitrification is feasible in small animal models. Based on our current achievements we intend to develop protocols for limb cryopreservation in large animals, aiming to advance the field of cryobiology towards the ambitious goal of human organ banking.