Infusion of human mesenchymal stromal cells during normothermic machine perfusion of porcine kidneys: a randomized, blinded, preclinical study

Infusion of human mesenchymal stromal cells during normothermic machine perfusion of porcine kidneys: a randomized, blinded, preclinical study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Infusion of human mesenchymal stromal cells during normothermic machine perfusion of porcine kidneys: a randomized, blinded, preclinical study Margaux Navez, Nicholas Gilbo, Morgan Vandermeulen, Pauline Erpicum, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8308681/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Kidney transplantation from donation after circulatory death (DCD) donors is increasingly used but remains associated with warm ischemic injury. Normothermic machine perfusion (NMP) enables functional assessment and therapeutic interventions. Mesenchymal stromal cells (MSCs) display immunomodulatory and regenerative properties, yet their translational efficacy during NMP remains unclear. Methods In a porcine model, six pairs of kidneys subjected to 30 minutes of warm ischemia followed by 3 hours of static cold storage underwent 6 hours of NMP. In each pair, one kidney randomly received an intra-arterial injection of placebo, while the contralateral kidney received 10 million clinical-grade human bone marrow–derived MSCs (hMSCs). Perfusion characteristics, glomerular filtration, tissue injury, and inflammatory markers were assessed. Results hMSC infusion during NMP was technically feasible and hemodynamically well tolerated, with no adverse effects on perfusion stability. Perfusion parameters, urine output, and creatinine/iohexol clearance showed no significant differences between groups. NGAL and cytokines levels increased during perfusion, but hMSCs did not alter their dynamics. Conclusions Intra-arterial delivery of clinical grade hMSCs during NMP was safe but did not improve renal function or reduce histological injury. These results highlight the challenges of achieving MSC engraftment during ex situ perfusion and highlight the need for refined strategies such as repeated dosing, prolonged perfusion, or extracellular vesicle therapy. Despite the negative findings, this study presents a highly translational large-animal model, supporting further investigation of MSC-based therapies in human kidneys discarded for transplantation. Mesenchymal Stromal Cell Transplantation Kidney Machine Perfusion Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Kidney transplantation remains the optimal treatment for end-stage renal disease; however, its potential is constrained by a significant global shortfall of suitable organ donors. 1 This shortage contributes to a prolonged transplantation waiting lists, and has driven a growing reliance on grafts obtained from donation after circulatory death (DCD) donors. 2 – 4 These DCD donors may increase organ availability for transplantation. 5 Evidence suggests that kidneys from DCD donors can achieve short- and mid-term survival rates comparable to those obtained from donation after brain death (DBD) donors. However, the higher incidence of primary graft dysfunction, coupled with the detrimental effects of the warm ischemia time inherent to DCD procurement, remains a significant limitation. 6 , 7 Machine perfusion is emerging as a valuable approach to kidney graft preservation, offering advantages over static cold storage (SCS), the current gold standard, by enabling both improvement and assessment of graft quality prior to transplantation. 8 – 12 In particular, normothermic machine perfusion (NMP) offers significant advantages to overcome current limitations in organ quality and preservation time. This technology provides enhanced preservation conditions, extends viable storage duration, enables kidney quality assessment, facilitates organ reconditioning, and allows for targeted therapeutic interventions. 13 – 15 NMP holds promise for clinical benefits, potentially improving early graft function, reducing ischemia–reperfusion injury, and enabling viability assessment prior to transplantation. 16 – 18 Recently, regenerative medicine has emerged within the field of organ transplantation, particularly in conjunction with machine perfusion techniques. 19 , 20 A potentially therapeutic intervention during NMP is the administration of mesenchymal stromal cells (MSCs), known for their immunomodulatory, anti-inflammatory, and regenerative properties. 21 – 23 We and others have previously investigated the use of MSCs in the setting of liver and kidney transplantation in early-phase clinical studies, demonstrating their favorable tolerability profile. 24 – 27 Building on this background, the present study investigates the potential therapeutic effects of clinical-grade human mesenchymal stem cells (hMSCs) in a large animal model. Specifically, we examine hMSCs perfused into renal grafts during NMP with the goal of mitigating ischemia-reperfusion injury. 28 The primary objective was to assess the feasibility of delivering hMSCs to ischemic porcine kidneys during NMP. Secondary objectives included evaluating their impact on perfusion quality, severity of renal tissue injury, modulation of key inflammatory markers, and overall kidney function. 2. Material and Methods 2.1. Ethics and Animals This study was designed and reported in accordance with the ARRIVE guidelines 2.0 for reporting animal research. Female laboratory pigs (30–60 kg), resulting from a Landrace and Pietrain cross, were sourced from the Walloon Agricultural Research Centre (CRA-W farm, Gembloux, Belgium). These animals were used for the collection of washed red blood cells and the procurement of kidneys. The study protocol was reviewed and approved by the Animal Ethics Committee of the University of Liege (approval number 21-2447). No formal sample size calculation was performed; six animals were deemed sufficient to assess feasibility and biological effects in this randomized, blinded, preclinical large-animal model, in accordance with the principles of animal reduction and refinement. Animal care and handling complied with the guidelines set out in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and the National Academy of Sciences, ensuring ethical and humane treatment throughout the experimental process. 2.2. Study Design Six pairs of kidneys were perfused simultaneously using NMP, following the protocol recently published by our group. 29 Following a 30-minute period of warm ischemia, kidneys underwent 3 hours of SCS, after which they were subjected to 6 hours of NMP. At 45 minutes of NMP, an intra-arterial injection was administered: kidneys were randomly assigned to receive either placebo (NMP group) or 10 million clinical-grade bone marrow-derived hMSCs (NMP + hMSC group). Perfusate and urine samples were collected every 30 minutes and every hour, respectively, during the 6-hour perfusion period. Renal tissue biopsies were obtained at the end of the NMP period ( Fig. 1 ). 2.3. MSCs and Placebo Preparation Detailed methodologies for clinical-grade hMSC culture have been reported previously. 28 In summary, hMSCs used in this study were produced under clinical-grade conditions by the Laboratory of Cell and Gene Therapy (LTCG), a GMP-licensed of accredited facility at the University Hospital of Liege. hMSCs were isolated from bone marrow aspirates of healthy allogeneic donors and processed via automated Ficoll-based mononuclear cell isolation closed-system. Cells were expanded in culture up to passage 2 using serum-containing media supplemented with gamma-irradiated foetal bovine serum. At passage 2, cells were harvested, formulated in a cryopreservation medium containing saline, human albumin, and 10% dimethyl sulfoxide, then frozen using a controlled-rate protocol before storage in the vapor phase of liquid nitrogen. For this study, hMSCs were thawed and directly diluted with a saline–albumin solution prior to intra-arterial administration during NMP. The final cell product was perfused within one hour post-thawing to ensure stability and viability. 28 The placebo consisted of a 20 mL syringe containing saline, human albumin and DMSO, identical in appearance and volume to the syringe containing 10 million thawed hMSCs, prepared as described above. 2.4. Randomization For each kidney pair, one kidney was randomly assigned to receive a placebo treatment (NMP group), while the contralateral kidney received the hMSC-based therapy (NMP + hMSC group). Randomization was performed by the LTCG, which was responsible for preparing and supplying both the placebo and hMSC products. On the day of the experiment, two identically prepared opaque syringes, labelled ‘right’ and ‘left’, were provided and administered to the corresponding kidneys in a blinded manner under standardized experimental conditions. To ensure blinded and unbiased evaluation, the surgical team, as well as all personnel involved in kidney graft procurement, NMP, sample collection, and data analysis, remained blinded to group allocation throughout the entire study. 2.5. Surgical Procedure Operative procedures have been described previously. 29 Briefly, animals were sedated thirty minutes prior to surgery via intramuscular injection of tiletamine/zolazepam (4.4 mg/kg; Zoletil 100, Virbac, Leuven, Belgium). An auricular vein catheter was placed, followed by intravenous induction of anesthesia and orotracheal intubation. Anesthesia was maintained using continuous propofol infusion (2–4 mg/kg/h; Aspen Pharma, Dublin, Ireland), boluses of sufentanyl (0.2 µg/kg Viatris; Canonsburg, Pennsylvania, USA). Animals were mechanically ventilated to maintain PaO₂ >100 mmHg and PaCO₂ between 35–45 mmHg. A carotid arterial catheter was inserted for hemodynamic monitoring and blood sampling. After intravenous administration of 25,000 IU of heparin, the abdominal vessels were cannulated. Warm ischemia was induced by clamping the thoracic aorta and maintained for 30 minutes. Subsequently, the kidneys were flushed in situ via the aortic cannula with 2 L of cold (4°C) IGL-1 preservation solution (Institut Georges Lopez, Lissieu, France), and the abdominal cavity was cooled with 4°C saline and topical slush ice. Whole blood was collected via the vena cava cannula during the flush and processed using a cell-saving system (Xtra autotransfusion system; LivaNova, Zaventem, Belgium) to obtain leukocyte-depleted, concentrated red blood cells. Animals underwent euthanasia by exsanguination, resulting in cardiac arrest under general anaesthesia. Following bilateral nephrectomy, the renal artery and the ureter were dissected and cannulated. Kidneys were then preserved by SCS in IGL-1 solution at 4°C for 3 hours. Prior to connection to the NMP circuit, each kidney was weighed and flushed on the back table with 500 mL of cold IGL-1 solution. 2.6. Normothermic Machine Perfusion As described in a previous study by our group, both kidneys from the same pig underwent simultaneous 6-hour NMP using two identical and independent, pressure-controlled, pulsatile perfusion systems. These were custom-made devices adapted from two discontinued clinical cardiopulmonary extracorporeal circulation systems (Stockert GmbH, Freiburg, Germany). 29 Each NMP circuit consisted of a kidney receptacle, roller pump, membrane oxygenator (Dideco Perfusion Tubing Systems; LivaNova, Zaventem, Belgium), and heat exchanger (Stockert GmbH, Freiburg, Germany) connected to coated tubing (LivaNova; Zaventem, Belgium). The arterial pressure (WPI, Friedberg, Germany) and flow rates (Sonotec, Halle, Germany) were continuously measured. The renal artery was cannulated and connected to the NMP circuit, while the renal vein was left open to drain into the circuit for reperfusion. The ureter was cannulated for urine collection. The NMP circuits were primed with a solution of autologous red blood cells and isotonic electrolyte solution (Plasmalyte; Baxter, Lessines, Belgium) to achieve a haematocrit of 20–30%. Flolan (Epoprostenol sodium, 7.5 µg/h; GSK, Wavre, Belgium) was continuously infused. Urine output was replaced 1:1 with Plasmalyte, and a parenteral nutrition solution (Aminomix 2 Novum; Fresenius Kabi, Willebroek, Belgium) was added to maintain glucose levels > 100 mg/dL. Sodium bicarbonate (10 mL, 8.4%; B Braun Melsungen, Germany) was used to maintain a pH in the range of 7.2 to 7.5. Perfusion temperature was maintained at 38°C with an oxygen flow of 100 mL/min at FiO₂ 21%. During perfusion, arterial pressure was gradually increased to a target range of 60–80 mmHg. Intrarenal resistance (IRR) was calculated as the ratio of arterial pressure to flow and expressed in mmHg/mL/min/100 g of tissue. 2.7. Perfusate and Tissue Collection A sampling line was connected to the arterial inflow cannula for perfusate collection. Samples of 2 mL were drawn into SST BD Microtainer tubes (BD, New Jersey, USA), centrifuged at 2000 × g for 5 minutes at 4°C. Supernatant was aliquoted and stored at − 80°C for subsequent analysis. Biochemical parameters (pH, sodium, potassium) were measured in the arterial line perfusate using the Epoc Blood Analysis System (Siemens Healthcare NV, Dilbeek, Belgium). Renal function was assessed via clearance of predefined concentrations of creatinine (75mg/L) and iohexol (518 mg/L) added to the perfusate during circuit priming (GE Healthcare, Diegem, Belgium). Clearance of these markers over time was used to evaluate excretory performance. The glomerular filtration rate was measured based on creatinine clearance and expressed as mL/6h/100g of kidney tissue. Perfusate and urine concentration of creatinine was determined at each sampling time point and cumulatively over the 6-hour NMP period to establish the filtration rate. All values were normalized to the kidney weight. At the end of perfusion, kidney biopsies were obtained for histological analysis. Tissue samples were fixed in 4% formalin, paraffin-embedded, and stained with haematoxylin-eosin and Periodic Acid–Schiff. Histopathological assessment was conducted by an experienced nephropathologist blinded to group allocation. The presence of hMSCs in porcine kidneys was evaluated by immunofluorescence on 5-µm sections using antibodies against human MHC class I (MHC Class I Antibody (JF10-38), NBP2-66946; Novus Biologicals) and porcine CD31 (CD31 (PECAM-1), (89C2), Mouse mAb, #3528; Cell Signaling). In addition, human-specific SRY and Alu sequences were targeted by PCR to confirm cellular localisation. All collected samples were analyzed, and no data points were excluded from the study. 2.8. ELISA Levels of neutrophil gelatinase-associated lipocalin (NGAL) were measured using commercially available enzyme-linked immunosorbent assay kits [human Lipocalin-2/NGAL Quantikine ELISA kits (R&D Systems, Minneapolis, MN, USA)]. A custom electrochemiluminescent Milliplex® assay was performed on perfusate samples to quantify cytokine expression. The 8-plex panel included IFN-Ɣ, IL-2, IL-10, IL-1⍺, IL-6, TNF-⍺, IL-1β and IL-8 (PCYTMAG-23K; Millipore, Darmstadt, Germany). All assays were carried out according to the manufacturer’s instructions. 2.9. Statistical Analysis A total of six animals (n = 6) were selected to account for expected biological variability in large-animal models. Results are reported as mean ± standard deviation. Data visualization was performed using GraphPad Prism (version 10). For comparative analyses, the area under the curve (AUC) was determined, followed by a non-parametric Wilcoxon signed-rank test. A generalized linear mixed model was used to assess the effects of time, treatment group (kidney), and their interaction, using a compound symmetry covariance structure to account for repeated measures. Assumptions of normality and homoscedasticity were verified, and log transformation was applied where appropriate. For two specific parameters, beta regression was used due to the distribution of the data. Model fit was evaluated using Akaike and Bayesian Information Criteria (AIC/BIC), with lower values indicating better fit. Statistical significance was defined as p < 0.05. All statistical analyses were conducted using SAS (version 9.4) and R (version 4.2.2), with the lnormimp package used for censored data imputation. 3. Results 3.1. Animal Characteristics Six pigs (mean weight: 40.50 ± 9.77 kg) underwent the experimental protocol enabling the procurement of six pairs of kidneys ( Table 1 ). In total, six kidneys were allocated to each group: the NMP group (1 right/5 left) and the NMP + hMSC group (5 right/1 left) (Suppl. Table 1). Baseline kidney weights were comparable between the control group (138.17 ± 32.46 g) and the treated group (134.83 ± 25.52 g; p = 0.56). At the end of perfusion, kidney weights had increased in both groups, with no significant difference observed in kidney weight at the end of perfusion (176.50 ± 45.92 g for NMP group vs. 184.33 ± 55.74 g for NMP + hMSC group; p = 0.31). Cold and warm ischemia times were identical between groups (CIT: 183.67 ± 7.61 min; WIT: 30 min). Baseline perfusate creatinine levels were also the same (1.04 ± 0.05 mg/dl in both groups). All kidneys were included in the final analysis. Table 1 Statistics of characteristics of paired kidneys included in the study (mean, SD and p-value) NMP group NMP + hMSCs group p-value Mean SD Mean SD Pig weight (kg) 40.50 9.77 40.50 9.77 - Pig creatinine, baseline (mg/dl) 1.04 0.05 1.04 0,05 - Kidney side (R/L) 1/5 - 5/1 - - Kidney weight, baseline (g) 138.17 32.46 134.83 25.52 0,56 Kidney weight, end perfusion (g) 176.50 45.92 184.33 55.74 0,31 CIT (min) 183.67 7,61 183.67 761 - WIT (min) 30.00 - 30.00 - - Note : CIT, cold ischemia time ; WIT, warm ischemia time 3.2. Perfusion Characteristics The administration of hMSCs did not significantly affect the majority of perfusion or biochemical parameters during the 6-hour perfusion period compared with controls (Suppl. Table 2). Electrolyte and acid–base homeostasis did not differ between groups, with comparable values for pH, sodium, potassium, pCO₂, and pO₂ (all p > 0.1) ( Fig. 2 ). Similarly, no significant intergroup differences were detected in glucose, lactate, creatinine, or iohexol clearance. In contrast, the NMP + hMSC group demonstrated significantly higher hematocrit (18.29 ± 2.73 vs. 17.40 ± 2.92; p = 0.018) and hemoglobin concentrations (6.21 ± 0.91 g/dl vs. 5.92 ± 0.98 g/dl; p = 0.017). In addition, mean perfusion pressure was greater in the NMP + hMSC group (53.65 ± 27.58 mmHg vs. 46.43 ± 16.51 mmHg; p = 0.035), while cumulative urine output was slightly lower (20.49 ± 15.68 ml vs. 21.70 ± 24.49 ml; p = 0.033). No significant difference in vascular resistance was observed between groups (3.28 ± 4.32 vs. 1.96 ± 2.24; p = 0.20). Although hMSC-treated kidneys exhibited a tendency towards greater weight gain (38.33 g ± 17.77 g for NMP group vs. 49.50 g ± 33.22 g for NMP + hMSC group), this difference was not statistically significant (p = 0.44) (Fig. 3 ). 3.3. Renal Function and Histology Clearance of creatinine and iohexol from the perfusate was used to evaluate glomerular filtration. Both analytes exhibited similar elimination profiles in the NMP + hMSC and NMP groups, with no significant differences observed in the calculated AUC. Only five kidneys (n = 5) were included in the GFR (Glomerular Filtration Rate) analysis, as one kidney pair from the same pig (pair 3) did not produce urine and was therefore excluded from this measurement. GFR followed a similar pattern, with slightly higher mean values in the NMP + hMSC group (108.30 ± 89.14 mL/6h/100g for NMP vs. 131.10 ± 74.12 mL/6h/100g for NMP + hMSC), though without statistical significance (p > 0.1). Histological evaluation of the kidney biopsies was conducted solely at the end of perfusion using PAS staining. Findings demonstrated features consistent with acute tubular injury, including loss of the proximal tubular brush border, thinning of the tubular epithelium and vacuolization of tubular cells. Mild peritubular capillarity was consistently observed across all samples (Fig. 4 ). Although hMSCs were administered intra-arterially, their detection in porcine renal tissue was inconclusive, and their presence could not be confirmed by immunofluorescence or PCR. 3.4. NGAL Dynamics Perfusate concentrations of NGALs increased significantly from baseline to 6 hours in both groups, consistent with progressive injury or stress during NMP. However, no significant intergroup differences were detected at any time point, indicating that hMSC treatment did not alter the temporal pattern of NGAL release (Fig. 5 ). 3.5. Cytokine Measurement No significant differences were observed between the NMP + hMSC group and NMP group for any of the measured cytokines. Nevertheless, a characteristic temporal pattern was noted across both groups: TNF-α concentrations peaked at 3 hours before declining towards baseline, while IL-6 and IL-10, initially nearly undetectable at the onset of perfusion, began to rise after 3-hours, suggesting a delayed cytokine response during NMP. IL-1α and IL-1β showed increased levels only at the 6-hour mark of perfusion. IL-2 remained below the detection threshold throughout the perfusion period (Fig. 6 ). 4. Discussion In this porcine model of NMP, intra-arterial infusion of 10 million clinical‐grade human bone marrow–derived MSCs was technically feasible and hemodynamically well tolerated, with no disruption of perfusion hemodynamic or adverse events. Despite seemingly efficient delivery, hMSC treatment failed to improve renal function (as assessed by creatinine clearance and urine output) nor did it reduce ischemia/reperfusion–induced histological injury when compared with controls. These findings are broadly consistent with previous reports from other groups. Lohmann et al. administered a comparable dose of MSCs into porcine kidneys during 4h of NMP and observed no functional or histological improvement after transplantation, despite confirming the presence of MSCs in renal tissue. 30 Pool et al . conducted two relevant studies: in 2020, they perfused ischemic pig kidneys with hMSCs for seven hours, resulting in reduced injury markers and increased growth factor release, yet without improvements in perfusion flow or excretory function 31 . In a 2019 study, the same group demonstrated that most MSCs rapidly disappeared from circulation, with only a limited number of cells retained in glomerular capillaries. 32 Our inability to detect MSCs post-NMP mirrors these findings, reinforcing the conclusion that cell engraftment during ex situ perfusion is minimal. A key methodological distinction between our study and that of Lohmann et al. lies in the preservation strategy: while their protocol incorporated a phase of cold oxygenated hypothermic perfusion (oxHMP) prior to NMP, our model utilized SCS followed immediately by NMP. Importantly, the warm and cold ischaemia times used in our study closely mirror those encountered in human DCD donor kidneys, suggesting that this may represent one of the more translationally relevant large-animal models described to date. The translational potential of MSC therapy in kidney transplantation has also been explored by our group in both preclinical and clinical settings, evaluating the anti-inflammatory, immunoregulatory, and reparative properties of MSCs in models of ischemia–reperfusion and transplantation. 24 , 26 In a pilot clinical trial, Erpicum et al. administered allogeneic bone marrow-derived MSCs (~ 2×10 6 cells/kg) into kidney transplant recipients and observed improved early graft function and increased regulatory T cell populations, without any adverse events. 25 These findings confirmed the safety of hMSC administration in humans and highlighted the potential of MSC-based therapies to enhance post-transplant recovery, although their long-term effects remain to be defined. Notably, donor-specific anti-HLA antibodies (DSAs) were detected in these studies, highlighting the risk of recipient sensitization following direct MSC injection. 25 , 33 In this context, NMP offers a strategy to deliver MSCs directly to the graft while theoretically minimizing the risk of recipient immunization. A key strength of our study lies in the use of clinical grade, hMSCs, manufactured under validated and standardized conditions in accordance with International Society for Cellular Therapy (ISCT) criteria. Unlike other preclinical studies that have employed research-grade, we deliberately selected the same hMSCs that have already been administered to patients in phase I/II clinical trials. 25 This strategy maximizes the reproducibility and translational relevance of our findings. Furthermore, with an experimental design that replicates the most recently developed preservation pathways (static cold storage followed by NMP), our study provides a unique and robust model for evaluating MSC-based interventions in a setting that most closely mirrors clinical reality. Nevertheless, several limitations must be acknowledged. Our inability to detect MSCs in the kidney following NMP, whether by immunofluorescence or PCR-based methods, highlights the challenge of tracking cells during perfusion. This observation suggests that the vast majority of MSCs are rapidly cleared or degraded, consistent with previous reports. 32 Although limited by a small sample size inherent to large-animal models, our design using paired kidneys minimized inter-individual variability. Moreover, the consistent absence of effects across functional, histological, and biomarker endpoints strongly suggests that a clinically meaningful benefit of a single MSCs dose during NMP is unlikely. Finally, the lack of standardized protocols in the literature—regarding MSC source, dose, timing, and administration route—remains a major obstacle to inter-study comparisons and reproducibility. This variability underscores the urgent need for harmonized protocols to generate robust and comparable results. In conclusion, while the safety and feasibility of hMSC delivery during NMP were confirmed, the lack of functional benefit provides critical information for refining future strategies and preventing unnecessary clinical translation of ineffective protocols. Our findings, together with prior reports 30 , 31 , indicate that refinements such as repeated dosing, prolonged perfusion, or the use of MSC-derived extracellular vesicles may be necessary to achieve consistent benefits. 34,35 Most importantly, by employing clinical-grade hMSCs under ischemia and preservation conditions comparable to current clinical practice, this study provides the most translationally relevant large-animal model to date. It thereby contributes valuable insights to guide further studies exploring MSC-based therapies in discarded human kidneys and to inform subsequent steps toward potential clinical translation. Future studies should prioritize strategies to enhance MSC persistence and functional activity, including repeated or prolonged dosing, pre-conditioning of cells, or the use of MSC-derived extracellular vesicles. Harmonization of experimental protocols across centers will be essential to enable direct comparability and accelerate clinical translation. List of abbreviations AUC, Area Under the Curve DBD, Donation After Brain Death DCD, Donation After Circulatory Death GFR, Glomerular Filtration Rate GMP, Good Manufacturing Practice hMSCs, Human Mesenchymal Stromal Cells IRR, Intrarenal Resistance LTCG, Laboratory of Cell and Gene Therapy MSCs, Mesenchymal Stromal Cells NGAL, Neutrophil Gelatinase-Associated Lipocalin NMP, Normothermic Machine Perfusion SCS, Static Cold Storage Declarations Ethics approval: This study was reviewed and approved by the Animal Ethics Committee of the University of Liege (approval number 21-2447) under the approved project entitled “Effects of normothermic perfusion of porcine kidneys with porcine mesenchymal stromal cells (MSCs) on ischemia–reperfusion injury”, approved on March 28, 2022. Animal care and handling complied with the guidelines set out in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and the National Academy of Sciences, ensuring ethical and humane treatment throughout the experimental process. Human mesenchymal stromal cells were obtained from donors who provided written informed consent for the use of their biological samples for research purposes, in accordance with institutional and national regulations. The use of Human MSCs was reviewed and approved by the Hospital–Faculty Ethics Committee of the University of Liège under the approved project entitled “TB0202F11-Consent-collecte moelle allo MSC”, with approval reference number 2025-470, approved on November 7, 2024. Consent for publication: Not applicable Availability of data and materials: All data analysed during this study are included in this published article and its supplementary files. The datasets used and analysed during the current study are available from the corresponding author on reasonable request. Competing interests: The authors declare that they have no competing interests. Funding: The authors received financial support for the research from grant obtained by the FNRS. A full list of collaborators is included in the Acknowledgements section. Authors’ Contributions: M.N., F.J., and O.D. designed the study. M.N., N.G., M.V., P.E., T.P.C., M.G.L., C.M, C.G, G.T., C.L., A.B., E.B, C.L.G., E.C., and O.D. performed the surgeries and experiments. M.N., N.G., F.J., and O.D. contributed to the analysis and interpretation of results. M.N. conducted statistical analyses. M.N., N.G., F.J., and O.D. were involved in data discussion and revised the manuscript for intellectual content. M.N. and O.D. participated in the writing of the paper. M.N. prepared the figures and tables. All authors have reviewed the results and approved the final version of the manuscript. Acknowledgments: The authors would like to thank the University Hospital Center for Biostatistics and Research Methods (B-STAT), and particularly Nadia Dardenne, for their assistance with the statistical data, and Jean-Paul Cheramy-Bien for technical support. We are also grateful to Pauline Van Delft and Lucie Detry for their valuable contributions to this project during their internships. This study was supported by grants; however, the funding sources not been involved in the design, execution, analysis or interpretation of the research. M.N. is the recipient of a Research Training in Industry and Agriculture (FRIA) grant. N.G., T.P.C., and F.J. are research fellows of the Fonds de la Recherche Scientifique—FNRS, which supported this work under Grants n° J.0069.22 and T.0095.24. Additional support was provided by the University of Liege (Crédit Forfaitaire de Fonctionnement, Fonds Léon Fredericq). Disclosure: The authors declare no conflicts of interest. 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The emergence of regenerative medicine in organ transplantation: 1st European Cell Therapy and Organ Regeneration Section meeting. Transpl Int. 2020;33(8):833–40. 10.1111/tri.13608 . Thompson ER, Bates L, Ibrahim IK, et al. Novel delivery of cellular therapy to reduce ischemia reperfusion injury in kidney transplantation. Am J Transpl. 2021;21(4):1402–14. Erpicum P, Detry O, Weekers L, et al. Mesenchymal stromal cell therapy in conditions of renal ischaemia/reperfusion. Nephrol Dialysis Transplantation. 2014;29(8):1487–93. 10.1093/ndt/gft538 . Sierra Parraga JM, Rozenberg K, Eijken M, et al. Effects of normothermic machine perfusion conditions on mesenchymal stromal cells. Front Immunol. 2019;10(APR):1–11. 10.3389/fimmu.2019.00765 . Perico N, Casiraghi F, Remuzzi G. Clinical Translation of Mesenchymal Stromal Cell Therapies in Nephrology. J Am Soc Nephrol. 2018;29(2):362–75. 10.1681/ASN.2017070781 . Detry O, Vandermeulen M, Delbouille MH, et al. Infusion of mesenchymal stromal cells after deceased liver transplantation: A phase I-II, open-label, clinical study. J Hepatol. 2017;67(1):47–55. Erpicum P, Weekers L, Detry O, et al. Infusion of third-party mesenchymal stromal cells after kidney transplantation: a phase I-II, open-label, clinical study. Kidney Int. 2019;95(3):693–707. Vandermeulen M, Mohamed-Wais M, Erpicum P, et al. Infusion of Allogeneic Mesenchymal Stromal Cells After Liver Transplantation: A 5-Year Follow-Up. Liver Transpl. 2022;28(4):636–46. 10.1002/lt.26323 . Thompson M, Mei SHJ, Wolfe D, et al. Cell therapy with intravascular administration of mesenchymal stromal cells continues to appear safe: An updated systematic review and meta-analysis. EClinicalMedicine. 2020;19:100249. 10.1016/j.eclinm.2019.100249 . Lechanteur C, Briquet A, Giet O, Delloye O, Baudoux E, Beguin Y. Clinical-scale expansion of mesenchymal stromal cells: a large banking experience. J Transl Med. 2016;14(1):145. 10.1186/s12967-016-0892-y . Navez M, Gilbo N, Vandermeulen M, et al. Simultaneous Ex Situ Normothermic Perfusion of Paired Kidneys in Pigs. Artificial Organs . Published online May. 2025;2:aor15016. 10.1111/aor.15016 . Lohmann S, Pool MBF, Rozenberg KM, et al. Mesenchymal stromal cell treatment of donor kidneys during ex vivo normothermic machine perfusion: A porcine renal autotransplantation study. Am J Transplant. 2021;21(7):2348–59. 10.1111/ajt.16473 . Pool MBF, Vos J, Eijken M, et al. Treating Ischemically Damaged Porcine Kidneys with Human Bone Marrow- A nd Adipose Tissue-Derived Mesenchymal Stromal Cells during Ex Vivo Normothermic Machine Perfusion. Stem Cells Dev. 2020;29(20):1320–30. 10.1089/scd.2020.0024 . Pool M, Eertman T, Parraga JS, et al. Infusing mesenchymal stromal cells into porcine kidneys during normothermic machine perfusion: Intact MSCs can be traced and localised to Glomeruli. Int J Mol Sci. 2019;20(14). 10.3390/ijms20143607 . Bezstarosti S, Erpicum P, Maggipinto G, et al. Allogeneic mesenchymal stromal cell therapy in kidney transplantation: should repeated human leukocyte antigen mismatches be avoided? Front Genet. 2024;15:1436194. 10.3389/fgene.2024.1436194 . Blondeel J, Gilbo N, De Bondt S, Monbaliu D. Stem cell Derived Extracellular Vesicles to Alleviate ischemia-reperfusion Injury of Transplantable Organs. A Systematic Review. Stem Cell Rev Rep. 2023;19(7):2225–50. 10.1007/s12015-023-10573-7 . Rampino T, Gregorini M, Germinario G, et al. Extracellular Vesicles Derived from Mesenchymal Stromal Cells Delivered during Hypothermic Oxygenated Machine Perfusion Repair Ischemic/Reperfusion Damage of Kidneys from Extended Criteria Donors. Biology (Basel). 2022;11(3). 10.3390/biology11030350 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8308681","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":580524333,"identity":"38b4ce7b-f7f4-408e-813e-db25e881b22f","order_by":0,"name":"Margaux Navez","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYPCDCgYZNhDN2EC0ljMMPCRqYWxj4GEgpEW3/ezjFz/bDjOYs58xfPhz3mEePvb2iw8Yd9jg1GJ2Jt3MsrctjcGyJ8fYQHLbYR42njPFBoxn0nBrOZDGZsDbZsNgcCAtTcIQpEUiJ02CEWgvTi3nn7EZ/m2TYDA4/yz9R+IcoBb5N+k/GNv+49ZyI435MdiWG8nHGA42gGxhPwYMhwN4tDxjY5Y5l8ZjcOPxYcmGY+lAv+QwSySeScbjsDTmj2/KDssZnE9s/PijxlpOvv34ww8fd9jh1AIEbBJAggdJgMeAIQGfBgYG5g9oAuwP8GsYBaNgFIyCkQYASzJTvGcITs8AAAAASUVORK5CYII=","orcid":"","institution":"University of Liège","correspondingAuthor":true,"prefix":"","firstName":"Margaux","middleName":"","lastName":"Navez","suffix":""},{"id":580524334,"identity":"2b5fda82-0119-42f7-83b5-9d0063ae48ed","order_by":1,"name":"Nicholas Gilbo","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Nicholas","middleName":"","lastName":"Gilbo","suffix":""},{"id":580524339,"identity":"33fdd15b-2fb4-4564-8ed2-90acc6dccad1","order_by":2,"name":"Morgan Vandermeulen","email":"","orcid":"","institution":"Centre Hospitalier Universitaire de Liège","correspondingAuthor":false,"prefix":"","firstName":"Morgan","middleName":"","lastName":"Vandermeulen","suffix":""},{"id":580524341,"identity":"ff446304-de26-40cb-af5e-65358e85c06e","order_by":3,"name":"Pauline Erpicum","email":"","orcid":"","institution":"Centre Hospitalier Universitaire de Liège","correspondingAuthor":false,"prefix":"","firstName":"Pauline","middleName":"","lastName":"Erpicum","suffix":""},{"id":580524343,"identity":"2fea04e2-9f95-4fc1-a7ef-ac9294d3a27d","order_by":4,"name":"Marc Gilbert Lagny","email":"","orcid":"","institution":"Centre Hospitalier Universitaire de Liège","correspondingAuthor":false,"prefix":"","firstName":"Marc","middleName":"Gilbert","lastName":"Lagny","suffix":""},{"id":580524345,"identity":"51953e8c-c81b-4716-b4d4-ca68d154b3ca","order_by":5,"name":"Clarisse Malisoux","email":"","orcid":"","institution":"Centre Hospitalier Universitaire de Liège","correspondingAuthor":false,"prefix":"","firstName":"Clarisse","middleName":"","lastName":"Malisoux","suffix":""},{"id":580524346,"identity":"d58da412-e71d-4a59-adb0-8f159339e1f6","order_by":6,"name":"Claire Guarin","email":"","orcid":"","institution":"Centre Hospitalier Universitaire de Liège","correspondingAuthor":false,"prefix":"","firstName":"Claire","middleName":"","lastName":"Guarin","suffix":""},{"id":580524347,"identity":"188e3c7c-80cd-4581-96bf-ac790de88ad3","order_by":7,"name":"Tiago Pinto Coelho","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Tiago","middleName":"Pinto","lastName":"Coelho","suffix":""},{"id":580524348,"identity":"eae1427e-ca8c-44f6-94a6-258d035040cb","order_by":8,"name":"Gabriel Thierry","email":"","orcid":"","institution":"Centre Hospitalier Universitaire de Liège","correspondingAuthor":false,"prefix":"","firstName":"Gabriel","middleName":"","lastName":"Thierry","suffix":""},{"id":580524349,"identity":"adb083c2-3012-4093-87a9-30df730db30f","order_by":9,"name":"Chantal Lechanteur","email":"","orcid":"","institution":"Centre Hospitalier Universitaire de Liège","correspondingAuthor":false,"prefix":"","firstName":"Chantal","middleName":"","lastName":"Lechanteur","suffix":""},{"id":580524350,"identity":"b6d51495-e4a4-4573-b61c-52ed555e0d78","order_by":10,"name":"Alexandra Briquet","email":"","orcid":"","institution":"Centre Hospitalier Universitaire de Liège","correspondingAuthor":false,"prefix":"","firstName":"Alexandra","middleName":"","lastName":"Briquet","suffix":""},{"id":580524351,"identity":"70cc96d1-2197-4afb-91d9-a41f04e5ba03","order_by":11,"name":"Etienne Baudoux","email":"","orcid":"","institution":"Centre Hospitalier Universitaire de Liège","correspondingAuthor":false,"prefix":"","firstName":"Etienne","middleName":"","lastName":"Baudoux","suffix":""},{"id":580524352,"identity":"c3190c49-60d7-4f4f-b4f7-c943c43410f8","order_by":12,"name":"Caroline Le Goff","email":"","orcid":"","institution":"Centre Hospitalier Universitaire de Liège","correspondingAuthor":false,"prefix":"","firstName":"Caroline","middleName":"Le","lastName":"Goff","suffix":""},{"id":580524353,"identity":"a06e2623-c2ce-4172-9662-2e46c48028a3","order_by":13,"name":"Etienne Cavalier","email":"","orcid":"","institution":"Centre Hospitalier Universitaire de Liège","correspondingAuthor":false,"prefix":"","firstName":"Etienne","middleName":"","lastName":"Cavalier","suffix":""},{"id":580524354,"identity":"bc676869-95d5-4a2a-84fa-74f9b03f891b","order_by":14,"name":"Jo Caers","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Jo","middleName":"","lastName":"Caers","suffix":""},{"id":580524355,"identity":"93485115-31b0-4477-91ae-142f91fe9475","order_by":15,"name":"François Jouret","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"François","middleName":"","lastName":"Jouret","suffix":""},{"id":580524356,"identity":"2651157b-499c-4545-8214-e423c8884cac","order_by":16,"name":"Olivier Detry","email":"","orcid":"","institution":"University of Liège","correspondingAuthor":false,"prefix":"","firstName":"Olivier","middleName":"","lastName":"Detry","suffix":""}],"badges":[],"createdAt":"2025-12-08 14:23:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8308681/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8308681/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101299443,"identity":"4182e59c-af00-4f62-b732-b91dfc0b0387","added_by":"auto","created_at":"2026-01-28 09:42:05","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":279616,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental design and samplings at different timings. Samples of urine and perfusate were collected during NMP. Biopsies were performed at the end of NMP. NMP, normothermic machine perfusion; T, timing; WIT, warm ischemia time; SCS, static cold storage; hMSCs, human mesenchymal stromal cells.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8308681/v1/47e1af99ceacd660a75d8109.jpeg"},{"id":101299277,"identity":"6d1db6d5-e252-4923-a9c9-7019c7b7613c","added_by":"auto","created_at":"2026-01-28 09:41:28","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":284889,"visible":true,"origin":"","legend":"\u003cp\u003ePerfusion characteristics during 6 hours of NMP with administration of placebo or 10 million hMSCs. (A) pH ;(B) sodium levels (mmol/l) ;(C) potassium levels (mmol/l) ;(D) arterial pressure (mmHg). Data presented as mean ± SD.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8308681/v1/8fa4d124f8e409f77b3607fb.jpeg"},{"id":101299489,"identity":"b7fc9cbc-de04-41c4-a028-63cafc742772","added_by":"auto","created_at":"2026-01-28 09:42:27","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":274188,"visible":true,"origin":"","legend":"\u003cp\u003ePerfusion characteristics during 6 hours of NMP with administration of placebo or 10 hMSCs. (A) Arterial flow (ml/min/100g) ;(B) Intrarenal Resistance (mmHg/ml/min) ;(C) Cumulative urine (ml/100g) ;(D) AUC analysis of flow; (E) AUC analysis of intrarenal resistance; (F) AUC analysis of cumulative urine and (G) weight gain (g). Data presented as mean ± SD.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8308681/v1/1df7516eeab36b6d282d7191.jpeg"},{"id":101299513,"identity":"0297d075-345e-45b5-ad90-a6cb71e173ab","added_by":"auto","created_at":"2026-01-28 09:43:07","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":32030,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of renal function during 6 hours of NMP with administration of placebo or 10 million hMSCs. (A) Elimination of creatinine in perfusate (mg/dl/100g) ;(B) Elimination of iohexol in perfusate (mg/dl/100g). Left panels: kinetics and right panels: AUC analyses. (C) Glomerular filtration rate (ml/6h/100g). \u0026nbsp;(D) Histological representation of kidney biopsies, original magnification x20, PAS staining. Data presented as mean ± SD.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8308681/v1/c702db48c3c3f4f14d1eb30c.jpg"},{"id":101299499,"identity":"77d0d66a-4eb7-4328-9d8e-ec2d9102203c","added_by":"auto","created_at":"2026-01-28 09:42:38","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":135506,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of kidney injuries during 6 hours of NMP with administration of placebo or 10 million hMSCs. (A) Concentration of NGAL in serum (ng/ml) ;(B) Concentration of NGAL in urine (ng/ml). Data presented as mean ± SD.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8308681/v1/05064d8fd63c1a96b8aa84dc.jpeg"},{"id":101299421,"identity":"a83fcd80-3e98-4fa2-90f6-a146ca746db7","added_by":"auto","created_at":"2026-01-28 09:41:56","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":32216,"visible":true,"origin":"","legend":"\u003cp\u003eConcentration of cytokines in NMP perfusates: IFNƔ, IL-10, IL-1⍺, IL-6, TNF⍺, IL-1β and IL-8. Left panels: kinetics and right panels: AUC analyses. Data presented as mean ± SD.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8308681/v1/f3722e4b1ec9c32dc249e635.jpg"},{"id":102962213,"identity":"b7a0b549-dc7c-44cb-a539-d7e08b2c805c","added_by":"auto","created_at":"2026-02-19 04:05:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1862607,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8308681/v1/0e147042-c599-4b1f-b896-23c982956a66.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Infusion of human mesenchymal stromal cells during normothermic machine perfusion of porcine kidneys: a randomized, blinded, preclinical study","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eKidney transplantation remains the optimal treatment for end-stage renal disease; however, its potential is constrained by a significant global shortfall of suitable organ donors.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e This shortage contributes to a prolonged transplantation waiting lists, and has driven a growing reliance on grafts obtained from donation after circulatory death (DCD) donors.\u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e These DCD donors may increase organ availability for transplantation.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Evidence suggests that kidneys from DCD donors can achieve short- and mid-term survival rates comparable to those obtained from donation after brain death (DBD) donors. However, the higher incidence of primary graft dysfunction, coupled with the detrimental effects of the warm ischemia time inherent to DCD procurement, remains a significant limitation.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eMachine perfusion is emerging as a valuable approach to kidney graft preservation, offering advantages over static cold storage (SCS), the current gold standard, by enabling both improvement and assessment of graft quality prior to transplantation.\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e In particular, normothermic machine perfusion (NMP) offers significant advantages to overcome current limitations in organ quality and preservation time. This technology provides enhanced preservation conditions, extends viable storage duration, enables kidney quality assessment, facilitates organ reconditioning, and allows for targeted therapeutic interventions.\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e NMP holds promise for clinical benefits, potentially improving early graft function, reducing ischemia\u0026ndash;reperfusion injury, and enabling viability assessment prior to transplantation.\u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eRecently, regenerative medicine has emerged within the field of organ transplantation, particularly in conjunction with machine perfusion techniques.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e A potentially therapeutic intervention during NMP is the administration of mesenchymal stromal cells (MSCs), known for their immunomodulatory, anti-inflammatory, and regenerative properties.\u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e We and others have previously investigated the use of MSCs in the setting of liver and kidney transplantation in early-phase clinical studies, demonstrating their favorable tolerability profile.\u003csup\u003e\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eBuilding on this background, the present study investigates the potential therapeutic effects of clinical-grade human mesenchymal stem cells (hMSCs) in a large animal model. Specifically, we examine hMSCs perfused into renal grafts during NMP with the goal of mitigating ischemia-reperfusion injury.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e The primary objective was to assess the feasibility of delivering hMSCs to ischemic porcine kidneys during NMP. Secondary objectives included evaluating their impact on perfusion quality, severity of renal tissue injury, modulation of key inflammatory markers, and overall kidney function.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Ethics and Animals\u003c/h2\u003e \u003cp\u003eThis study was designed and reported in accordance with the ARRIVE guidelines 2.0 for reporting animal research. Female laboratory pigs (30\u0026ndash;60 kg), resulting from a Landrace and Pietrain cross, were sourced from the Walloon Agricultural Research Centre (CRA-W farm, Gembloux, Belgium). These animals were used for the collection of washed red blood cells and the procurement of kidneys. The study protocol was reviewed and approved by the Animal Ethics Committee of the University of Liege (approval number 21-2447). No formal sample size calculation was performed; six animals were deemed sufficient to assess feasibility and biological effects in this randomized, blinded, preclinical large-animal model, in accordance with the principles of animal reduction and refinement. Animal care and handling complied with the guidelines set out in the \u003cem\u003eGuide for the Care and Use of Laboratory Animals\u003c/em\u003e published by the National Institutes of Health and the National Academy of Sciences, ensuring ethical and humane treatment throughout the experimental process.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Study Design\u003c/h2\u003e \u003cp\u003eSix pairs of kidneys were perfused simultaneously using NMP, following the protocol recently published by our group.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Following a 30-minute period of warm ischemia, kidneys underwent 3 hours of SCS, after which they were subjected to 6 hours of NMP. At 45 minutes of NMP, an intra-arterial injection was administered: kidneys were randomly assigned to receive either placebo (NMP group) or 10\u0026nbsp;million clinical-grade bone marrow-derived hMSCs (NMP\u0026thinsp;+\u0026thinsp;hMSC group). Perfusate and urine samples were collected every 30 minutes and every hour, respectively, during the 6-hour perfusion period. Renal tissue biopsies were obtained at the end of the NMP period \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. MSCs and Placebo Preparation\u003c/h2\u003e \u003cp\u003eDetailed methodologies for clinical-grade hMSC culture have been reported previously.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e In summary, hMSCs used in this study were produced under clinical-grade conditions by the Laboratory of Cell and Gene Therapy (LTCG), a GMP-licensed of accredited facility at the University Hospital of Liege. hMSCs were isolated from bone marrow aspirates of healthy allogeneic donors and processed via automated Ficoll-based mononuclear cell isolation closed-system. Cells were expanded in culture up to passage 2 using serum-containing media supplemented with gamma-irradiated foetal bovine serum. At passage 2, cells were harvested, formulated in a cryopreservation medium containing saline, human albumin, and 10% dimethyl sulfoxide, then frozen using a controlled-rate protocol before storage in the vapor phase of liquid nitrogen. For this study, hMSCs were thawed and directly diluted with a saline\u0026ndash;albumin solution prior to intra-arterial administration during NMP. The final cell product was perfused within one hour post-thawing to ensure stability and viability.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e The placebo consisted of a 20 mL syringe containing saline, human albumin and DMSO, identical in appearance and volume to the syringe containing 10\u0026nbsp;million thawed hMSCs, prepared as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Randomization\u003c/h2\u003e \u003cp\u003eFor each kidney pair, one kidney was randomly assigned to receive a placebo treatment (NMP group), while the contralateral kidney received the hMSC-based therapy (NMP\u0026thinsp;+\u0026thinsp;hMSC group). Randomization was performed by the LTCG, which was responsible for preparing and supplying both the placebo and hMSC products. On the day of the experiment, two identically prepared opaque syringes, labelled \u0026lsquo;right\u0026rsquo; and \u0026lsquo;left\u0026rsquo;, were provided and administered to the corresponding kidneys in a blinded manner under standardized experimental conditions. To ensure blinded and unbiased evaluation, the surgical team, as well as all personnel involved in kidney graft procurement, NMP, sample collection, and data analysis, remained blinded to group allocation throughout the entire study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Surgical Procedure\u003c/h2\u003e \u003cp\u003eOperative procedures have been described previously.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Briefly, animals were sedated thirty minutes prior to surgery via intramuscular injection of tiletamine/zolazepam (4.4 mg/kg; Zoletil 100, Virbac, Leuven, Belgium). An auricular vein catheter was placed, followed by intravenous induction of anesthesia and orotracheal intubation. Anesthesia was maintained using continuous propofol infusion (2\u0026ndash;4 mg/kg/h; Aspen Pharma, Dublin, Ireland), boluses of sufentanyl (0.2 \u0026micro;g/kg Viatris; Canonsburg, Pennsylvania, USA). Animals were mechanically ventilated to maintain PaO₂ \u0026gt;100 mmHg and PaCO₂ between 35\u0026ndash;45 mmHg. A carotid arterial catheter was inserted for hemodynamic monitoring and blood sampling. After intravenous administration of 25,000 IU of heparin, the abdominal vessels were cannulated. Warm ischemia was induced by clamping the thoracic aorta and maintained for 30 minutes. Subsequently, the kidneys were flushed in situ via the aortic cannula with 2 L of cold (4\u0026deg;C) IGL-1 preservation solution (Institut Georges Lopez, Lissieu, France), and the abdominal cavity was cooled with 4\u0026deg;C saline and topical slush ice. Whole blood was collected via the vena cava cannula during the flush and processed using a cell-saving system (Xtra autotransfusion system; LivaNova, Zaventem, Belgium) to obtain leukocyte-depleted, concentrated red blood cells. Animals underwent euthanasia by exsanguination, resulting in cardiac arrest under general anaesthesia. Following bilateral nephrectomy, the renal artery and the ureter were dissected and cannulated. Kidneys were then preserved by SCS in IGL-1 solution at 4\u0026deg;C for 3 hours. Prior to connection to the NMP circuit, each kidney was weighed and flushed on the back table with 500 mL of cold IGL-1 solution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Normothermic Machine Perfusion\u003c/h2\u003e \u003cp\u003eAs described in a previous study by our group, both kidneys from the same pig underwent simultaneous 6-hour NMP using two identical and independent, pressure-controlled, pulsatile perfusion systems. These were custom-made devices adapted from two discontinued clinical cardiopulmonary extracorporeal circulation systems (Stockert GmbH, Freiburg, Germany).\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Each NMP circuit consisted of a kidney receptacle, roller pump, membrane oxygenator (Dideco Perfusion Tubing Systems; LivaNova, Zaventem, Belgium), and heat exchanger (Stockert GmbH, Freiburg, Germany) connected to coated tubing (LivaNova; Zaventem, Belgium). The arterial pressure (WPI, Friedberg, Germany) and flow rates (Sonotec, Halle, Germany) were continuously measured. The renal artery was cannulated and connected to the NMP circuit, while the renal vein was left open to drain into the circuit for reperfusion. The ureter was cannulated for urine collection. The NMP circuits were primed with a solution of autologous red blood cells and isotonic electrolyte solution (Plasmalyte; Baxter, Lessines, Belgium) to achieve a haematocrit of 20\u0026ndash;30%. Flolan (Epoprostenol sodium, 7.5 \u0026micro;g/h; GSK, Wavre, Belgium) was continuously infused. Urine output was replaced 1:1 with Plasmalyte, and a parenteral nutrition solution (Aminomix 2 Novum; Fresenius Kabi, Willebroek, Belgium) was added to maintain glucose levels\u0026thinsp;\u0026gt;\u0026thinsp;100 mg/dL. Sodium bicarbonate (10 mL, 8.4%; B Braun Melsungen, Germany) was used to maintain a pH in the range of 7.2 to 7.5. Perfusion temperature was maintained at 38\u0026deg;C with an oxygen flow of 100 mL/min at FiO₂ 21%. During perfusion, arterial pressure was gradually increased to a target range of 60\u0026ndash;80 mmHg. Intrarenal resistance (IRR) was calculated as the ratio of arterial pressure to flow and expressed in mmHg/mL/min/100 g of tissue.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Perfusate and Tissue Collection\u003c/h2\u003e \u003cp\u003eA sampling line was connected to the arterial inflow cannula for perfusate collection. Samples of 2 mL were drawn into SST BD Microtainer tubes (BD, New Jersey, USA), centrifuged at 2000 \u0026times; g for 5 minutes at 4\u0026deg;C. Supernatant was aliquoted and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent analysis. Biochemical parameters (pH, sodium, potassium) were measured in the arterial line perfusate using the Epoc Blood Analysis System (Siemens Healthcare NV, Dilbeek, Belgium). Renal function was assessed via clearance of predefined concentrations of creatinine (75mg/L) and iohexol (518 mg/L) added to the perfusate during circuit priming (GE Healthcare, Diegem, Belgium). Clearance of these markers over time was used to evaluate excretory performance. The glomerular filtration rate was measured based on creatinine clearance and expressed as mL/6h/100g of kidney tissue. Perfusate and urine concentration of creatinine was determined at each sampling time point and cumulatively over the 6-hour NMP period to establish the filtration rate. All values were normalized to the kidney weight. At the end of perfusion, kidney biopsies were obtained for histological analysis. Tissue samples were fixed in 4% formalin, paraffin-embedded, and stained with haematoxylin-eosin and Periodic Acid\u0026ndash;Schiff. Histopathological assessment was conducted by an experienced nephropathologist blinded to group allocation. The presence of hMSCs in porcine kidneys was evaluated by immunofluorescence on 5-\u0026micro;m sections using antibodies against human MHC class I (MHC Class I Antibody (JF10-38), NBP2-66946; Novus Biologicals) and porcine CD31 (CD31 (PECAM-1), (89C2), Mouse mAb, #3528; Cell Signaling). In addition, human-specific SRY and Alu sequences were targeted by PCR to confirm cellular localisation. All collected samples were analyzed, and no data points were excluded from the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. ELISA\u003c/h2\u003e \u003cp\u003eLevels of neutrophil gelatinase-associated lipocalin (NGAL) were measured using commercially available enzyme-linked immunosorbent assay kits [human Lipocalin-2/NGAL Quantikine ELISA kits (R\u0026amp;D Systems, Minneapolis, MN, USA)]. A custom electrochemiluminescent Milliplex\u0026reg; assay was performed on perfusate samples to quantify cytokine expression. The 8-plex panel included IFN-Ɣ, IL-2, IL-10, IL-1⍺, IL-6, TNF-⍺, IL-1β and IL-8 (PCYTMAG-23K; Millipore, Darmstadt, Germany). All assays were carried out according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Statistical Analysis\u003c/h2\u003e \u003cp\u003eA total of six animals (n\u0026thinsp;=\u0026thinsp;6) were selected to account for expected biological variability in large-animal models. Results are reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Data visualization was performed using GraphPad Prism (version 10). For comparative analyses, the area under the curve (AUC) was determined, followed by a non-parametric Wilcoxon signed-rank test. A generalized linear mixed model was used to assess the effects of time, treatment group (kidney), and their interaction, using a compound symmetry covariance structure to account for repeated measures. Assumptions of normality and homoscedasticity were verified, and log transformation was applied where appropriate. For two specific parameters, beta regression was used due to the distribution of the data. Model fit was evaluated using Akaike and Bayesian Information Criteria (AIC/BIC), with lower values indicating better fit. Statistical significance was defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All statistical analyses were conducted using SAS (version 9.4) and R (version 4.2.2), with the \u003cem\u003elnormimp\u003c/em\u003e package used for censored data imputation.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Animal Characteristics\u003c/h2\u003e \u003cp\u003eSix pigs (mean weight: 40.50\u0026thinsp;\u0026plusmn;\u0026thinsp;9.77 kg) underwent the experimental protocol enabling the procurement of six pairs of kidneys \u003cb\u003e(\u003c/b\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e In total, six kidneys were allocated to each group: the NMP group (1 right/5 left) and the NMP\u0026thinsp;+\u0026thinsp;hMSC group (5 right/1 left) \u003cb\u003e(Suppl. Table\u0026nbsp;1).\u003c/b\u003e Baseline kidney weights were comparable between the control group (138.17\u0026thinsp;\u0026plusmn;\u0026thinsp;32.46 g) and the treated group (134.83\u0026thinsp;\u0026plusmn;\u0026thinsp;25.52 g; p\u0026thinsp;=\u0026thinsp;0.56). At the end of perfusion, kidney weights had increased in both groups, with no significant difference observed in kidney weight at the end of perfusion (176.50\u0026thinsp;\u0026plusmn;\u0026thinsp;45.92 g for NMP group vs. 184.33\u0026thinsp;\u0026plusmn;\u0026thinsp;55.74 g for NMP\u0026thinsp;+\u0026thinsp;hMSC group; p\u0026thinsp;=\u0026thinsp;0.31). Cold and warm ischemia times were identical between groups (CIT: 183.67\u0026thinsp;\u0026plusmn;\u0026thinsp;7.61 min; WIT: 30 min). Baseline perfusate creatinine levels were also the same (1.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 mg/dl in both groups). All kidneys were included in the final analysis.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStatistics of characteristics of paired kidneys included in the study (mean, SD and p-value)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eNMP group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eNMP\u0026thinsp;+\u0026thinsp;hMSCs group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSD\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePig weight (kg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePig creatinine, baseline (mg/dl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0,05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKidney side (R/L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1/5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5/1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKidney weight, baseline (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e138.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e32.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e134.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKidney weight, end perfusion (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e176.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e184.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e55.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCIT (min)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e183.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7,61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e183.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e761\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWIT (min)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003cem\u003eNote : CIT, cold ischemia time ; WIT, warm ischemia time\u003c/em\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Perfusion Characteristics\u003c/h2\u003e \u003cp\u003eThe administration of hMSCs did not significantly affect the majority of perfusion or biochemical parameters during the 6-hour perfusion period compared with controls \u003cb\u003e(Suppl. Table\u0026nbsp;2).\u003c/b\u003e Electrolyte and acid\u0026ndash;base homeostasis did not differ between groups, with comparable values for pH, sodium, potassium, pCO₂, and pO₂ (all p\u0026thinsp;\u0026gt;\u0026thinsp;0.1) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e Similarly, no significant intergroup differences were detected in glucose, lactate, creatinine, or iohexol clearance. In contrast, the NMP\u0026thinsp;+\u0026thinsp;hMSC group demonstrated significantly higher hematocrit (18.29\u0026thinsp;\u0026plusmn;\u0026thinsp;2.73 vs. 17.40\u0026thinsp;\u0026plusmn;\u0026thinsp;2.92; p\u0026thinsp;=\u0026thinsp;0.018) and hemoglobin concentrations (6.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91 g/dl vs. 5.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98 g/dl; p\u0026thinsp;=\u0026thinsp;0.017). In addition, mean perfusion pressure was greater in the NMP\u0026thinsp;+\u0026thinsp;hMSC group (53.65\u0026thinsp;\u0026plusmn;\u0026thinsp;27.58 mmHg vs. 46.43\u0026thinsp;\u0026plusmn;\u0026thinsp;16.51 mmHg; p\u0026thinsp;=\u0026thinsp;0.035), while cumulative urine output was slightly lower (20.49\u0026thinsp;\u0026plusmn;\u0026thinsp;15.68 ml vs. 21.70\u0026thinsp;\u0026plusmn;\u0026thinsp;24.49 ml; p\u0026thinsp;=\u0026thinsp;0.033). No significant difference in vascular resistance was observed between groups (3.28\u0026thinsp;\u0026plusmn;\u0026thinsp;4.32 vs. 1.96\u0026thinsp;\u0026plusmn;\u0026thinsp;2.24; p\u0026thinsp;=\u0026thinsp;0.20). Although hMSC-treated kidneys exhibited a tendency towards greater weight gain (38.33 g\u0026thinsp;\u0026plusmn;\u0026thinsp;17.77 g for NMP group vs. 49.50 g\u0026thinsp;\u0026plusmn;\u0026thinsp;33.22 g for NMP\u0026thinsp;+\u0026thinsp;hMSC group), this difference was not statistically significant (p\u0026thinsp;=\u0026thinsp;0.44) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Renal Function and Histology\u003c/h2\u003e \u003cp\u003eClearance of creatinine and iohexol from the perfusate was used to evaluate glomerular filtration. Both analytes exhibited similar elimination profiles in the NMP\u0026thinsp;+\u0026thinsp;hMSC and NMP groups, with no significant differences observed in the calculated AUC. Only five kidneys (n\u0026thinsp;=\u0026thinsp;5) were included in the GFR (Glomerular Filtration Rate) analysis, as one kidney pair from the same pig (pair 3) did not produce urine and was therefore excluded from this measurement. GFR followed a similar pattern, with slightly higher mean values in the NMP\u0026thinsp;+\u0026thinsp;hMSC group (108.30\u0026thinsp;\u0026plusmn;\u0026thinsp;89.14 mL/6h/100g for NMP vs. 131.10\u0026thinsp;\u0026plusmn;\u0026thinsp;74.12 mL/6h/100g for NMP\u0026thinsp;+\u0026thinsp;hMSC), though without statistical significance (p\u0026thinsp;\u0026gt;\u0026thinsp;0.1). Histological evaluation of the kidney biopsies was conducted solely at the end of perfusion using PAS staining. Findings demonstrated features consistent with acute tubular injury, including loss of the proximal tubular brush border, thinning of the tubular epithelium and vacuolization of tubular cells. Mild peritubular capillarity was consistently observed across all samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Although hMSCs were administered intra-arterially, their detection in porcine renal tissue was inconclusive, and their presence could not be confirmed by immunofluorescence or PCR.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4. NGAL Dynamics\u003c/h2\u003e \u003cp\u003ePerfusate concentrations of NGALs increased significantly from baseline to 6 hours in both groups, consistent with progressive injury or stress during NMP. However, no significant intergroup differences were detected at any time point, indicating that hMSC treatment did not alter the temporal pattern of NGAL release (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Cytokine Measurement\u003c/h2\u003e \u003cp\u003eNo significant differences were observed between the NMP\u0026thinsp;+\u0026thinsp;hMSC group and NMP group for any of the measured cytokines. Nevertheless, a characteristic temporal pattern was noted across both groups: TNF-α concentrations peaked at 3 hours before declining towards baseline, while IL-6 and IL-10, initially nearly undetectable at the onset of perfusion, began to rise after 3-hours, suggesting a delayed cytokine response during NMP. IL-1α and IL-1β showed increased levels only at the 6-hour mark of perfusion. IL-2 remained below the detection threshold throughout the perfusion period (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this porcine model of NMP, intra-arterial infusion of 10\u0026nbsp;million clinical‐grade human bone marrow\u0026ndash;derived MSCs was technically feasible and hemodynamically well tolerated, with no disruption of perfusion hemodynamic or adverse events. Despite seemingly efficient delivery, hMSC treatment failed to improve renal function (as assessed by creatinine clearance and urine output) nor did it reduce ischemia/reperfusion\u0026ndash;induced histological injury when compared with controls.\u003c/p\u003e \u003cp\u003eThese findings are broadly consistent with previous reports from other groups. Lohmann \u003cem\u003eet al.\u003c/em\u003e administered a comparable dose of MSCs into porcine kidneys during 4h of NMP and observed no functional or histological improvement after transplantation, despite confirming the presence of MSCs in renal tissue.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Pool \u003cem\u003eet al\u003c/em\u003e. conducted two relevant studies: in 2020, they perfused ischemic pig kidneys with hMSCs for seven hours, resulting in reduced injury markers and increased growth factor release, yet without improvements in perfusion flow or excretory function \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. In a 2019 study, the same group demonstrated that most MSCs rapidly disappeared from circulation, with only a limited number of cells retained in glomerular capillaries.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Our inability to detect MSCs post-NMP mirrors these findings, reinforcing the conclusion that cell engraftment during \u003cem\u003eex situ\u003c/em\u003e perfusion is minimal. A key methodological distinction between our study and that of Lohmann \u003cem\u003eet al.\u003c/em\u003e lies in the preservation strategy: while their protocol incorporated a phase of cold oxygenated hypothermic perfusion (oxHMP) prior to NMP, our model utilized SCS followed immediately by NMP. Importantly, the warm and cold ischaemia times used in our study closely mirror those encountered in human DCD donor kidneys, suggesting that this may represent one of the more translationally relevant large-animal models described to date.\u003c/p\u003e \u003cp\u003eThe translational potential of MSC therapy in kidney transplantation has also been explored by our group in both preclinical and clinical settings, evaluating the anti-inflammatory, immunoregulatory, and reparative properties of MSCs in models of ischemia\u0026ndash;reperfusion and transplantation.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e In a pilot clinical trial, Erpicum \u003cem\u003eet al.\u003c/em\u003e administered allogeneic bone marrow-derived MSCs (~\u0026thinsp;2\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/kg) into kidney transplant recipients and observed improved early graft function and increased regulatory T cell populations, without any adverse events.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e These findings confirmed the safety of hMSC administration in humans and highlighted the potential of MSC-based therapies to enhance post-transplant recovery, although their long-term effects remain to be defined. Notably, donor-specific anti-HLA antibodies (DSAs) were detected in these studies, highlighting the risk of recipient sensitization following direct MSC injection.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e In this context, NMP offers a strategy to deliver MSCs directly to the graft while theoretically minimizing the risk of recipient immunization.\u003c/p\u003e \u003cp\u003eA key strength of our study lies in the use of clinical grade, hMSCs, manufactured under validated and standardized conditions in accordance with International Society for Cellular Therapy (ISCT) criteria. Unlike other preclinical studies that have employed research-grade, we deliberately selected the same hMSCs that have already been administered to patients in phase I/II clinical trials.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e This strategy maximizes the reproducibility and translational relevance of our findings. Furthermore, with an experimental design that replicates the most recently developed preservation pathways (static cold storage followed by NMP), our study provides a unique and robust model for evaluating MSC-based interventions in a setting that most closely mirrors clinical reality.\u003c/p\u003e \u003cp\u003eNevertheless, several limitations must be acknowledged. Our inability to detect MSCs in the kidney following NMP, whether by immunofluorescence or PCR-based methods, highlights the challenge of tracking cells during perfusion. This observation suggests that the vast majority of MSCs are rapidly cleared or degraded, consistent with previous reports.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Although limited by a small sample size inherent to large-animal models, our design using paired kidneys minimized inter-individual variability. Moreover, the consistent absence of effects across functional, histological, and biomarker endpoints strongly suggests that a clinically meaningful benefit of a single MSCs dose during NMP is unlikely. Finally, the lack of standardized protocols in the literature\u0026mdash;regarding MSC source, dose, timing, and administration route\u0026mdash;remains a major obstacle to inter-study comparisons and reproducibility. This variability underscores the urgent need for harmonized protocols to generate robust and comparable results.\u003c/p\u003e \u003cp\u003eIn conclusion, while the safety and feasibility of hMSC delivery during NMP were confirmed, the lack of functional benefit provides critical information for refining future strategies and preventing unnecessary clinical translation of ineffective protocols. Our findings, together with prior reports \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, indicate that refinements such as repeated dosing, prolonged perfusion, or the use of MSC-derived extracellular vesicles may be necessary to achieve consistent benefits. \u003csup\u003e34,35\u003c/sup\u003e Most importantly, by employing clinical-grade hMSCs under ischemia and preservation conditions comparable to current clinical practice, this study provides the most translationally relevant large-animal model to date. It thereby contributes valuable insights to guide further studies exploring MSC-based therapies in discarded human kidneys and to inform subsequent steps toward potential clinical translation. Future studies should prioritize strategies to enhance MSC persistence and functional activity, including repeated or prolonged dosing, pre-conditioning of cells, or the use of MSC-derived extracellular vesicles. Harmonization of experimental protocols across centers will be essential to enable direct comparability and accelerate clinical translation.\u003c/p\u003e"},{"header":"List of abbreviations","content":"\u003cp\u003eAUC, Area Under the Curve\u003c/p\u003e\n\u003cp\u003eDBD, Donation After Brain Death\u003c/p\u003e\n\u003cp\u003eDCD, Donation After Circulatory Death\u003c/p\u003e\n\u003cp\u003eGFR, Glomerular Filtration Rate\u003c/p\u003e\n\u003cp\u003eGMP, Good Manufacturing Practice\u003c/p\u003e\n\u003cp\u003ehMSCs, Human Mesenchymal Stromal Cells\u003c/p\u003e\n\u003cp\u003eIRR, Intrarenal Resistance\u003c/p\u003e\n\u003cp\u003eLTCG, Laboratory of Cell and Gene Therapy\u003c/p\u003e\n\u003cp\u003eMSCs, Mesenchymal Stromal Cells\u003c/p\u003e\n\u003cp\u003eNGAL, Neutrophil Gelatinase-Associated Lipocalin\u003c/p\u003e\n\u003cp\u003eNMP, Normothermic Machine Perfusion\u003c/p\u003e\n\u003cp\u003eSCS, Static Cold Storage\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval:\u0026nbsp;\u003c/strong\u003eThis study was reviewed and approved by the Animal Ethics Committee of the University of Liege (approval number 21-2447) under the approved project entitled “Effects of normothermic perfusion of porcine kidneys with porcine mesenchymal stromal cells (MSCs) on ischemia–reperfusion injury”, approved on March 28, 2022. Animal care and handling complied with the guidelines set out in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and the National Academy of Sciences, ensuring ethical and humane treatment throughout the experimental process. Human mesenchymal stromal cells were obtained from donors who provided written informed consent for the use of their biological samples for research purposes, in accordance with institutional and national regulations. The use of Human MSCs was reviewed and approved by the Hospital–Faculty Ethics Committee of the University of Liège under the approved project entitled “TB0202F11-Consent-collecte moelle allo MSC”, with approval reference number 2025-470, approved on November 7, 2024.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eAll data analysed during this study are included in this published article and its supplementary files. The datasets used and analysed during the current study are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The authors received financial support for the research from grant obtained by the FNRS. A full list of collaborators is included in the Acknowledgements section.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ Contributions:\u003c/strong\u003e M.N., F.J., and O.D.\u0026nbsp;designed the study. M.N., N.G., M.V., P.E., T.P.C., M.G.L., C.M, C.G, G.T., C.L., A.B., E.B, C.L.G., E.C., and O.D.\u0026nbsp;performed\u0026nbsp;the\u0026nbsp;surgeries and experiments. M.N., N.G., F.J., and O.D.\u0026nbsp;contributed to the analysis and interpretation of results. M.N. conducted statistical analyses. M.N., N.G., F.J., and O.D. were\u0026nbsp;involved in data discussion and revised the manuscript for intellectual content. M.N. and O.D. participated in the writing of the paper. M.N. prepared the figures and tables. All authors\u0026nbsp;have reviewed the results and approved the final version of the\u0026nbsp;manuscript. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e The authors would like to thank the University Hospital Center for Biostatistics and Research Methods (B-STAT), and particularly Nadia Dardenne, for their assistance with the statistical data, and Jean-Paul Cheramy-Bien for technical support. We are also grateful to Pauline Van Delft and Lucie Detry for their valuable contributions to this project during their internships. This study was supported by grants; however, the funding sources not been involved in the design, execution, analysis or interpretation of the research. M.N. is the recipient of a Research Training in Industry and Agriculture (FRIA) grant. N.G., T.P.C., and F.J. are research fellows of the Fonds de la Recherche Scientifique—FNRS, which supported this work under Grants n° J.0069.22 and T.0095.24. Additional support was provided by the University of Liege (Crédit Forfaitaire de Fonctionnement, Fonds Léon Fredericq).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflicts of interest. The authors declare that they have not use AI-generated work in this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWebster AC, Nagler EV, Morton RL, Masson P. Chronic Kidney Disease. 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Biology (Basel). 2022;11(3). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/biology11030350\u003c/span\u003e\u003cspan address=\"10.3390/biology11030350\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Mesenchymal Stromal Cell, Transplantation, Kidney, Machine Perfusion","lastPublishedDoi":"10.21203/rs.3.rs-8308681/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8308681/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eKidney transplantation from donation after circulatory death (DCD) donors is increasingly used but remains associated with warm ischemic injury. Normothermic machine perfusion (NMP) enables functional assessment and therapeutic interventions. Mesenchymal stromal cells (MSCs) display immunomodulatory and regenerative properties, yet their translational efficacy during NMP remains unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn a porcine model, six pairs of kidneys subjected to 30 minutes of warm ischemia followed by 3 hours of static cold storage underwent 6 hours of NMP. In each pair, one kidney randomly received an intra-arterial injection of placebo, while the contralateral kidney received 10\u0026nbsp;million clinical-grade human bone marrow\u0026ndash;derived MSCs (hMSCs). Perfusion characteristics, glomerular filtration, tissue injury, and inflammatory markers were assessed.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003ehMSC infusion during NMP was technically feasible and hemodynamically well tolerated, with no adverse effects on perfusion stability. Perfusion parameters, urine output, and creatinine/iohexol clearance showed no significant differences between groups. NGAL and cytokines levels increased during perfusion, but hMSCs did not alter their dynamics.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eIntra-arterial delivery of clinical grade hMSCs during NMP was safe but did not improve renal function or reduce histological injury. These results highlight the challenges of achieving MSC engraftment during \u003cem\u003eex situ\u003c/em\u003e perfusion and highlight the need for refined strategies such as repeated dosing, prolonged perfusion, or extracellular vesicle therapy. Despite the negative findings, this study presents a highly translational large-animal model, supporting further investigation of MSC-based therapies in human kidneys discarded for transplantation.\u003c/p\u003e","manuscriptTitle":"Infusion of human mesenchymal stromal cells during normothermic machine perfusion of porcine kidneys: a randomized, blinded, preclinical study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-28 09:08:46","doi":"10.21203/rs.3.rs-8308681/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dddeb9aa-6c18-4aaf-bfb6-852c403a6592","owner":[],"postedDate":"January 28th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-11T11:57:34+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-28 09:08:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8308681","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8308681","identity":"rs-8308681","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}