Exceeding the Limits of Static Cold Storage in Limb Transplantation Using Subnormothermic Machine Perfusion

Abstract

Background: For 50 years, static cold storage (SCS) has been the gold standard for solid organ preservation in transplantation. Although logistically convenient, this preservation method presents important constraints in terms of duration and cold ischemia-induced lesions. We aimed to develop a machine perfusion (MP) protocol for recovery of vascularized composite allografts (VCA) after static cold preservation and determine its effects in a rat limb transplantation model.

Methods: Partial hindlimbs were procured from Lewis rats and subjected to SCS in Histidine-Tryptophan-Ketoglutarate solution for 0, 12, 18, 24, and 48 hours. They were then either transplanted (Txp), subjected to subnormothermic machine perfusion (SNMP) for 3 hours with a modified Steen solution, or to SNMP + Txp. Perfusion parameters were assessed for blood gas and electrolytes measurement, and flow rate and arterial pressures were monitored continuously. Histology was assessed at the end of perfusion. For select SCS durations, graft survival and clinical outcomes after transplantation were compared between groups at 21 days.

Results: Transplantation of limbs preserved for 0, 12, 18, and 24-hour SCS resulted in similar survival rates at postoperative day 21. Grafts cold-stored for 48 hours presented delayed graft failure (p = 0.0032). SNMP of limbs after 12-hour SCS recovered the vascular resistance, potassium, and lactate levels to values similar to limbs that were not subjected to SCS. However, 18-hour SCS grafts developed significant edema during SNMP recovery. Transplantation of grafts that had undergone a mixed preservation method (12-hour SCS + SNMP + Txp) resulted in better clinical outcomes based on skin clinical scores at day 21 post-transplantation when compared to the SCS + Txp group (p = 0.01613).

Conclusion: To date, VCA MP is still limited to animal models and no protocols are yet developed for graft recovery. Our study suggests that ex vivo SNMP could help increase the preservation duration and limit cold ischemia-induced injury in VCA transplantation.

Fig. 1. Experimental design of the study.

Sixty animals were used (four donors and four recipients in each of the five groups of the SCS + Txp study = 40, four donors for each of the three groups of the perfusion studies [SCS + SNMP] = 12, four donors and four recipients in the SCS + SNMP + Txp study = 8).

Fig. 2

Machine perfusion set-up for ex vivo rat limb SNMP. (1) Roller pump (Masterflex). (2) Radnoti glass oxygenator. (3) Radnoti bubble trap. (4) Pressure sensor. (5) Pressure monitor. (6) Radnoti organ basin containing a rat hindlimb. (7) Sterile perfusate (Steen +). (8) Empty sterile bottle for outflow drainage. (9) Oxygen inflow (0.5 L/min). (10) Heater tubing containing sterile water at 21°C and circulating in the jacketed components (organ basin, bubble trap, and oxygenator).

Fig. 3

(A) Changes in VCA survival after 0 (control), 12, 18, 24, and 48-hour SCS (n = 4 in all groups). After 12, 18, or 24-hours of SCS, similar VCA survival percentages were obtained when compared to control transplantations (p >0.05). VCA failure was found significantly increased at the end of study in the 48-hour SCS group (p = 0.0032). (B) Post-transplant clinical evolution after 12, 18, 24, and 48-hour SCS. Epidermolysis leaves scarring tissue in the 24 and 48-hour SCS groups, preventing hair regrowth. VCA, vascularized composite allograft.

Fig. 4. Post-static cold storage perfusion parameters during a 3-hour SNMP using our modified Steen solution (n = 4 in each group).

Panel (A) shows high initial resistances after cold storage decreasing after 30 minutes (group 12-hour SCS) and 60 minutes in the 18-hour SCS group. Panel (B) shows a steady increase of the flowrate reaching 0.8 mL/min at the end of the recovery perfusion. Oxygen uptake (panel C) is lower after a period of SCS compared to fresh control. Potassium and lactate levels (D, E) steadily decreased during perfusion. Potassium release was significantly higher in the 12 (p = 0.003) and 18 hours SCS group (p = 0.0002). Edema (F) was significantly higher in the 18-hour SCS group compared to 12-hour SCS (p = 0.0238).

Fig. 5.. Pathologic evaluation of muscle biopsies.

Muscle biopsies after SCS, before and after SNMP (H&E, 400x). Focal myocyte necrosis (black arrows) was present after 12-hour SCS (A) and mild edema at 3-hour SNMP (B). After 18-hour SCS alone (C), there was diffuse myocyte breakdown with extensive myocyte injury and interstitial edema (asterisk) after 3-hour SNMP (D).

Fig. 6 (A–F). Clinical outcomes.

Recovering limbs using SNMP enhances graft quality and accelerate the healing of skin lesions due to extended cold ischemia.

Fig. 7. Pathologic evaluation of skin and muscle biopsies at POD21 (H&E, 400x).

In the 12-hour SCS group, the epidermis (A) showed focal necrosis and ulceration (*) while the muscle (C) showed myocyte injury and atrophy (encircled). The 3-hour SNMP group showed normal skin (B) while the muscle biopsy (D) showed mild interstitial edema.