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24-hour Supercooling Preservation Of Vascularized Composite Allografts On Rats: A Proof-of-concept Study
Pierre Tawa, MD, Marion Goutard, MD, Casie A. Pendexter, BSc, Alexandre G. Lellouch, MD, Curtis L. Cetrulos, Jr., MD, Korkut Uygun, PhD.
Massachusetts General Hospital, Boston, MA, USA.

PURPOSE:
Vascularized composite allotransplantation (VCA) remains the only alternative for patients suffering from severe disfigurements nonaddressable by conventional reconstructive surgery. However, ischemia time is still a major limiting factor considering irreversible muscular injury can occur after 6 hours of static cold storage (SCS). Organ preservation technologies have led to the development of storage protocols using subzero temperatures by combining cryopreservative agents (CPA) and machine perfusion, thus effectively extending rat liver’s ex vivo viable preservation up to 4 days. In this study, we aimed to optimize an extended subzero non-freezing (SZNF) preservation protocol for rat VCA.
METHODS:Eight partial hindlimbs were procured from Lewis male rats and perfused with subnormothermic machine perfusion (SNMP) with a modified Steen solution mixed with either 300 mM (n=5) or 100 mM (n=3) of 3-O’Methylglucose (3-OMG). Limbs were then cooled down to +4°C, flushed and submerged with a preservation solution (Histidine-tryptophan-ketoglutarate + Trehalose 50 mM + Glycerol 5% + Polyethylene glycol 5%) in a sealed sterile plastic bag. VCAs were supercooled for 24 hours at -4°C, then were rewarmed and recovered for 2 hours using SNMP. As controls, three limbs were cold stored at +4°C for 24 hours and subjected to 2-hr SNMP recovery (Figure 1). Perfusion parameters, electrolytes and blood gas were monitored during both loading and recovery phases. Muscle biopsies were collected at the end of the experiment for histological analysis.
RESULTS:
Both 300 and 100 mM groups displayed similar perfusion parameters at the end of loading SNMP. All limbs were successfully supercooled for 24 hours without ice nucleation. During recovery SNMP, the supercooling groups showed higher glucose consumption compared with the SCS control group, with statistical difference between the 300 mM group compared to SCS for the first 90 minutes. Resistances decreased faster in supercooled limbs compared with controls. Lactate release was lower in the 300 mM overtime, and potassium level drastically decreased in supercooled limbs while it remained high in the control group until the end of recovery (Figure 2). The 300 mM group showed more edema than 100 mM and SCS groups after recovery (+34.59% ± 21.67, +24.80% ± 0.69, and +13.24% ± 4.45, respectively). Histology showed limited cell injury after recovery in all groups, but important interstitial edema in both experimental groups.
CONCLUSION:
This study is a proof-of-concept that extending VCA ex-vivo preservation up to 24 hours with supercooling is feasible in a rodent model, with a three-phase protocol including CPA loading, subzero storage in a preservation solution and limb recovery with SNMP. Further optimization is still expected to allow for its application in a transplantation model, which would be a major breakthrough in the fields of VCA an organ preservation.




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