Abstract Purpose There are no established criteria for discontinuing ex vivo normothermic limb perfusion (EVNLP) before irreversible damage occurs. This study evaluates weight gain as an indicator of injury during EVNLP. Methods Sixteen Yorkshire pig forelimbs were procured and preserved using EVNLP with a hemoglobin‐based oxygen carrier (HBOC‐201) or static cold storage. EVNLP continued until termination criteria were met: arterial pressure ≥ 115 mm Hg, compartment pressure > 30 mm Hg, or 20% reduction of oxygen saturation. Limb weight, contractility, hemodynamics, perfusate electrolytes, metabolites and gases were recorded. Muscles were biopsied 6‐h, and muscle injury scores (MIS) calculated. Forearm compartment pressures and indocyanine green (ICG) angiography were recorded at endpoint. Outcomes were compared at 2%, 5%, 10%, and 20% limb weight gain. Results EVNLP lasted 20 ± 3 h. Weight gain was observed after 13 ± 5 h (2%), 15 ± 6 h (5%), 16 ± 6 h (10%), and 19 ± 4 h (20%). Weight correlated positively with MIS (ρ = 0.92, p < 0.0001), potassium (ρ = −1.00, p < 0.0001), pressure (ρ = 0.78, p < 0.0001), and negatively with contractility (ρ = −0.96, p = 0.011). At 5% weight gain, MIS (p < 0.0001), potassium (p = 0.03), and lactate (p < 0.0001) were significantly higher than baseline. Median muscle contractility was 5 [3–5] at 2% weight gain, 4 [1–5] at 5%, 3 [0–4] and 2 [0–2] at 10% and 20%, respectively. At 20% weight gain, contractility was significantly lower than baseline (p = 0.003). Percent weight gain correlated negatively with endpoint ICG hoof fluorescence (r = −0.712, p = 0.047). Conclusions Weight gain correlated with microscopic muscle injury and was the earliest evidence of limb dysfunction. Weight gain may serve as a criterion for discontinuation of EVNLP.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1111/aor.14442

Weight gain is an early indicator of injury in ex vivo normothermic limb perfusion (EVNLP)

Drugs that induce reversible slowing of metabolic and physiological processes would have great value for organ preservation, especially for organs with high susceptibility to hypoxia-reperfusion injury, such as the heart. Using whole-organism screening of metabolism, mobility, and development in Xenopus , we identified an existing drug, SNC80, that rapidly and reversibly slows biochemical and metabolic activities while preserving cell and tissue viability. Although SNC80 was developed as a delta opioid receptor activator, we discovered that its ability to slow metabolism is independent of its opioid modulating activity as a novel SNC80 analog (WB3) with almost 1,000 times less delta opioid receptor binding activity is equally active. Metabolic suppression was also achieved using SNC80 in microfluidic human organs-on-chips, as well as in explanted whole porcine hearts and limbs, demonstrating the cross-species relevance of this approach and potential clinical relevance for surgical transplantation. Pharmacological induction of physiological slowing in combination with organ perfusion transport systems may offer a new therapeutic approach for tissue and organ preservation for transplantation, trauma management, and enhancing patient survival in remote and low-resource locations. 

Identification of pharmacological inducers of a reversible hypometabolic state for whole organ preservation

The applications of Vascularized composite allotransplantation (VCA) are increasing since the first successful hand transplantation in 1998. However, the abundance of muscle tissue makes VCA's vulnerable to ischemia-reperfusion injury (IRI), which has detrimental effects on the outcome of the procedure, restricting allowable donor-to-recipient time and limiting its widespread use. The current clinical method is Static cold storage (SCS) and this allows only 6 h before irreversible damage occurs upon reperfusion. In order to overcome this obstacle, the focus of research has been shifted towards the prospect of ex-vivo perfusion preservation which already has an established clinical role in solid organ transplants especially in the last decade. In this comprehensive qualitative review, we compile the literature on all VCA machine perfusion models and we aim to highlight the essentials of an ex vivo perfusion set-up, the different strategies, and their associated outcomes.

Review of machine perfusion studies in vascularized composite allotransplant preservation

An injectable ‘biostasis’ drug that could slow organ injury by inducing a state of suspended animation through reversible slowing of metabolic processes would have great value for organ preservation and treatment of badly injured patients at the point-of-care. Using whole-organism screening of metabolism, mobility, and development in Xenopus, we identified an existing drug, SNC80, that rapidly and reversibly slows biochemical and metabolic activities while preserving cell and tissue viability, independently of its known delta opioid receptor modulating activity. Metabolic suppression was also achieved using SNC80 in cultured cells and microfluidic human organs-on-chips, as well as in explanted whole porcine hearts and limbs, demonstrating the cross-species relevance of this approach and potential clinical relevance for surgical transplantation. Thermal proteome profiling revealed that SNC80 targets the NCX1/EEAT1 membrane transport system and chemical modulation of NCX1 induces biostasis in non-hibernating Xenopus tadpoles. Molecular induction of biostasis may offer a new therapeutic approach for organ preservation, trauma management, and enhancing patient survival in remote and low-resource locations.

/pdf/identification-of-a-pharmaceutical-biostasis-inducer-that-1qs28p2b.pdf

Identification of a pharmaceutical biostasis inducer that slows metabolism in multiple vertebrates that do not hibernate

Background: Vascularized composite allotransplantation has become a viable reconstructive option for selected patients, but preservation remains a major barrier to broader clinical application. Static cold storage is the current gold standard, yet ischemia reperfusion injury and limited preservation times restrict its effectiveness. Recent advances in machine perfusion and subzero non-freezing storage (or supercooling) have prompted renewed interest in optimizing graft viability. Methods: Following PRISMA guidelines, we systematically searched PubMed, EMBASE, and Cochrane, covering studies published from June 2022 through August 2025 for studies on ex vivo preservation of vascularized composite allotransplantations. Eligible articles included original studies in English evaluating postharvest, pretransplant preservation strategies. Data extracted were study design, preservation methods, perfusates, and primary outcomes. Risk of bias was assessed using SYRCLE for animal studies and JBI for human/cadaver studies. Results: Seventeen studies met inclusion criteria: one on static cold storage, thirteen on machine perfusion, and three on supercooling. Static cold storage research has declined, with the only recent study investigating sub-normothermic machine perfusion as a recovery adjunct. Machine perfusion studies focused on optimization of perfusion parameters, perfusate composition, and circuit design. Red blood cell-based perfusates remained common, but alternative oxygen carriers such as polymerized hemoglobin-based oxygen carrier-201 and dextran oxygen microcarriers showed promise despite edema-related challenges. Supercooling studies demonstrated feasibility of multi-day preservation in rodent and porcine models. Overall, risk of bias was high or unclear across animal studies, mainly due to selection and performance bias, whereas the single human ex vivo study showed low risk of bias. Conclusions: The field of vascularized composite allograft preservation is expanding rapidly, with machine perfusion and supercooling emerging as the most promising strategies to extend graft viability beyond the limits of static cold storage. However, translation to clinical setting remains limited by small preclinical studies, methodological heterogeneity, and the paucity of functional and immunologic endpoints. Standardized protocols, robust large-animal models, and eventual human feasibility trials are needed to establish clinically applicable preservation strategies. Level of evidence: IV 

Preservation Strategies for Vascularized Composite Allotransplantation: An Updated Systematic Review of a Rapidly Expanding Field

Preclinical Evaluation of the VP.S ENCORE™ Cardiac Preservation Device

Novel Portable Hypothermic Perfusion Preservation Device Enhances Cardiac Output of Donated Human Hearts

Purpose: Heart transplantation is the gold standard therapy for patients with end-stage heart failure. However, the supply of donor hearts does not meet the demand, and many patients die on the waitlist. The purpose of this study was to test the efficacy of a novel machine perfusion device, the VP.S ENCORE® cardiac preservation device, in preserving porcine hearts conditioned with a biostasis-inducing compound (WC1) during prolonged 6-8 hour perfusion. Methods: Isolated porcine hearts were recovered under anesthesia. Following cannulation, the hearts were placed in the VP.S ENCORE® cardiac preservation device for oxygenated perfusion at 23-25°C for either 6 (n = 3) or 8 (n = 4) hours. At hour 3, WC1 (n = 2) was administered through the arterial line and the hearts continued to be perfused for additional 5 hours. Hourly flow, pressure, temperature, blood gases, and various metabolites were recorded. After preservation, hearts were placed in a Langendorff model for reperfusion and evaluated for cardiac function. Biopsy samples were taken before and at the end of preservation for histological and gene expression analyses. Results: During perfusion, oxygen consumption was significantly reduced in WC1-treated hearts (p < 0.0001). However, during re-perfusion, WC1-treated hearts had an initial lower oxygen consumption that recovered to normal with time and temperature. Before defibrillating the hearts, oxygen consumption was 1.61 ± 0.59 (vehicle) & 1.05 ± 0.39 (WC1), while after defibrillation the oxygen consumption increased to 3.29 ± 0.63 & 2.66 ± 0.32 ml/min/100g, respectively. The contractility of the WC1-treated hearts was similar to a normal human heart in vivo. Furthermore, gene expression analysis showed a significant reduction (p < 0.0008) of inflammation (IL-6, TNFα), hypoxia (Hif1a), & cell death markers (p53) in WC1 treated hearts when compared to control hearts. Conclusions: Our preliminary results demonstrate that the portable, simple-to-use VP.S ENCORE® cardiac preservation machine perfusion device coupled with the torpor properties of compounds like WC1 may provide a new paradigm to prolonged organ preservation, and may offer many advantages over currently tested machine perfusion technologies. Further, this innovative work is a result of integrating science and engineering to target unmet patients’ needs in the field of organ preservation.Figure 1. Subnormothermic Perfusion of Porcine Hearts using VP.S ENCORE® 

CARD28: Prolonged Graft Preservation of Biostasis Induced Porcine Hearts using the VP.S ENCORE® Machine Perfusion Device

Prolonged Preservation of Pediatric Size Hearts Using the VP.S ENCORE® Hypothermic Oxygenated Machine Perfusion Device

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