Since the 1960s, static cold storage (a cold organ in a box of ice) has been the mainstay in preservation. The worldwide shortage of organs has driven research in to the use of machine perfusion pre-implantation to both assess as well as optimise function. Machine Perfusion (MP) can either be cold or warm. Warm perfusion, usually at body temperature, is termed normothermic (NMP). Several machines for liver, kidneys, lungs and hearts exist in the clinical practise [9Balfoussia D, Yerrakalva D, Hamaoui K, Papalois V. Advances in machine perfusion graft viability assessment in kidney, liver, pancreas, lung, and heart transplant. Exp Clin Transplant 2012; 10(2): 87-100.
[http://dx.doi.org/10.6002/ect.2011.0167] ]. Continuous perfusion of the organ at body temperature ex-vivo, permits efficient oxygen delivery as well as essential nutrients in a closed circuit. The perfusate is renewed or filtered and in the case of renal ex-vivo NMP, the production of urine observed can be collected for analysis [10Hosgood SA. Renal transplantation after ex vivo normothermic perfusion: The first clinical study. Am J Transplant 2013; 13(5): 1246-52.]. Real time assessment of the organ during NMP can be made with clinical and biochemical parameters studied in order to “risk stratify” to outcome. Compared to static cold storage, a more sophisticated assessment can be made.

NMP devices have aided the expansion of Donation after Circulatory Death (DCD) organs that, due to the exposure of prolonged warm ischaemia, have conventionally been considered inferior to both living and brain dead donors [11Summers DM, Johnson RJ, Allen J, et al. Analysis of factors that affect outcome after transplantation of kidneys donated after cardiac death in the UK: A cohort study. Lancet 2010; 376(9749): 1303-11.
[http://dx.doi.org/10.1016/S0140-6736(10)60827-6] ]. The ability to machine perfuse under artificial physiological parameters, enables organ assessment prior to implantation.

Renal transplantation remains the treatment of choice for patients with End Stage Renal Disease (ESRD). DCD renal allografts have higher rates of Delayed Graft Function (DGF) as well as Primary Non Function (PNF) [11Summers DM, Johnson RJ, Allen J, et al. Analysis of factors that affect outcome after transplantation of kidneys donated after cardiac death in the UK: A cohort study. Lancet 2010; 376(9749): 1303-11.
[http://dx.doi.org/10.1016/S0140-6736(10)60827-6] ]. Early study into the use of ex-vivo NMP has been encouraging with significantly lower rates of DGF observed in machine perfused kidney allografts versus static cold storage [10Hosgood SA. Renal transplantation after ex vivo normothermic perfusion: The first clinical study. Am J Transplant 2013; 13(5): 1246-52.]. Long term survival outcomes from graft rejection as well as chronic allograft failure in the form of interstitial fibrosis lend the use of NMP towards stem cell therapy. To date there are no combined studies into the effect of renal allograft NMP and stem cell usage.

Mesenchymal stem cells are multipotent progenitors, which in current clinical practise are usually derived from Bone Marrow (BM). They have immunomodulatory and immune-evasive properties, making them suitable for cell based therapy. MSCs are known to activate T and B regulatory cells and interfere with maturation and activation of Antigen Presenting Cells (APCs) [2Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284(5411): 143-7.
[http://dx.doi.org/10.1126/science.284.5411.143] , 12Cho D-I, Kim MR, Jeong H-Y, et al. Mesenchymal stem cells reciprocally regulate the M1/M2 balance in mouse bone marrow-derived macrophages. Exp Mol Med 2014; 46(1): e470.
[http://dx.doi.org/10.1038/emm.2013.135] , 13Sotiropoulou PA, Perez SA, Gritzapis AD, Baxevanis CN, Papamichail M. Interactions between human mesenchymal stem cells and natural killer cells. Stem Cells 2006; 24(1): 74-85.
[http://dx.doi.org/10.1634/stemcells.2004-0359] ]. The potential use of MSCs is not solely limited to cellular repair and regeneration. Preclinical studies elucidated their immunoregulatory potential; however their clinical use still remains to be explored. Accumulated evidence showed MSC helps the development of tolerance through the induction of T and B regulatory cells and its inhibitory and modulatory effect on various immune effector cells including APCs, macrophages, neutrophils, mast cells, T and B cells also enhance the anti-inflammatory pathways and supress the cytotoxic activities of NK cells [2Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284(5411): 143-7.
[http://dx.doi.org/10.1126/science.284.5411.143] , 12Cho D-I, Kim MR, Jeong H-Y, et al. Mesenchymal stem cells reciprocally regulate the M1/M2 balance in mouse bone marrow-derived macrophages. Exp Mol Med 2014; 46(1): e470.
[http://dx.doi.org/10.1038/emm.2013.135] , 13Sotiropoulou PA, Perez SA, Gritzapis AD, Baxevanis CN, Papamichail M. Interactions between human mesenchymal stem cells and natural killer cells. Stem Cells 2006; 24(1): 74-85.
[http://dx.doi.org/10.1634/stemcells.2004-0359] ]. They produce wide range of Th2, anti-inflammatory [14Kyurkchiev D, Bochev I, Ivanova-Todorova E, et al. Secretion of immunoregulatory cytokines by mesenchymal stem cells. World J Stem Cells 2014; 6(5): 552-70.
[http://dx.doi.org/10.4252/wjsc.v6.i5.552] ] and ant apoptotic [15Galleu A, Riffo-Vasquez Y. Apoptosis in mesenchymal stromal cells induces in vivo recipient-mediated immunomodulation. Sci Transl Med 2017; 9(416) pii: eaam 7828.
[http://dx.doi.org/10.1126/scitranslmed.aam7828] ] effectors including TGF-B, HGF, NO, HO-1, IDO, TNF-TRAIL and PD-L1.

Preclinical studies established their safety and their effect on enhancing graft survival, diminishing rejection rate and lymphocyte proliferation [16Bartholomew A, Sturgeon C, Siatskas M, et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 2002; 30(1): 42-8.
[http://dx.doi.org/10.1016/S0301-472X(01)00769-X] -18Inoue S, Popp FC, Koehl GE, et al. Immunomodulatory effects of mesenchymal stem cells in a rat organ transplant model. Transplantation 2006; 81(11): 1589-95.
[http://dx.doi.org/10.1097/01.tp.0000209919.90630.7b] ]. However, early clinical study reports didn’t show similar potential yet due to various reasons including their use in conjunction with immunosuppression medications that already supress various function, the influence of microenvironments and the immunogenicity when allogenic - MSC used [18Inoue S, Popp FC, Koehl GE, et al. Immunomodulatory effects of mesenchymal stem cells in a rat organ transplant model. Transplantation 2006; 81(11): 1589-95.
[http://dx.doi.org/10.1097/01.tp.0000209919.90630.7b] -21Giai Via A, Frizziero A, Oliva F. Biological properties of mesenchymal stem cells from different sources. Muscles Ligaments Tendons J 2012; 2(3): 154-62.].

A recent study by Pan et al, demonstrated similar 2 year survival outcomes of patients given low dose Calcineurin Inhibitor (CNI) with donor derived BM-MSCs compared with standard CNI use alone [4Pan GH, Chen Z, Xu L, et al. Low-dose tacrolimus combined with donor-derived mesenchymal stem cells after renal transplantation: A prospective, non-randomized study. Oncotarget 2016; 7(11): 12089-101.
[http://dx.doi.org/10.18632/oncotarget.7725] ]. The authors do not give a formal explanation for the mechanism of action and the method of MSC immunosuppression is not completely understood. Early in vitro work has suggested that autologous T-cell proliferation is inhibited by MSCs through the secretion of both transforming growth factor-β [22Di Nicola M, Carlo-Stella C, Magni M, et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood 2002; 99(10): 3838-43.
[http://dx.doi.org/10.1182/blood.V99.10.3838] ]. Indeed some authors have suggested that because of the production of extracellular vesicles, such as exosomes, as well as cytokines and growth factors, MSC immunomodulation is related to the trophic and stimulatory function over the cellular differentiation aspect [23Ankrum JA, Ong JF, Karp JM. Mesenchymal stem cells: Immune evasive, not immune privileged. Nat Biotechnol 2014; 32(3): 252-60.
[http://dx.doi.org/10.1038/nbt.2816] ]. Both low levels of Major Histocompatibility Complex (MHC) class I & II as well as CD-40 expression further support this hypothesis [23Ankrum JA, Ong JF, Karp JM. Mesenchymal stem cells: Immune evasive, not immune privileged. Nat Biotechnol 2014; 32(3): 252-60.
[http://dx.doi.org/10.1038/nbt.2816] ]. This is, however, contradictory to previously held belief that BM-MSCs do not express MHC class II or their co-stimulatory molecules including CD-40 [24Ryan JM, Barry FP, Murphy JM, Mahon BP. Mesenchymal stem cells avoid allogeneic rejection. J Inflamm (Lond) 2005; 2: 8.
[http://dx.doi.org/10.1186/1476-9255-2-8] ].

Ischaemia Reperfusion Injury (IRI), is the development of organ dysfunction following arterial clamp release and subsequent perfusion of the allograft. Reactive oxygen species’ (ROS) development at the time of IRI is a leading contributor DCD renal allograft dysfunction [25Waller HL, Harper SJF, Hosgood SA, et al. Biomarkers of oxidative damage to predict ischaemia-reperfusion injury in an isolated organ perfusion model of the transplanted kidney. Free Radic Res 2006; 40(11): 1218-25.
[http://dx.doi.org/10.1080/10715760600907368] ]. The role of MSCs in ameliorating IRI, remains unclear. Casiraghi et al have studied the topic in-vivo and in-vitro. The group postulated that the presence of MSCs engraft during the reperfusion process, induces complement activation as well as expression of pro-inflammatory cytokines, which contribute to the subsequent organ dysfunction [26Casiraghi F, Azzollini N, Todeschini M, et al. Localization of mesenchymal stromal cells dictates their immune or proinflammatory effects in kidney transplantation. Am J Transplant 2012; 12(9): 2373-83.
[http://dx.doi.org/10.1111/j.1600-6143.2012.04115.x] ]. They hypothesized that MSCs could be directed towards acquisition of pro-inflammatory functions when exposed to specific mediators of inflammation such as ROS during renal IRI. This non NMP work, however, contrasts that found in the only clinical study of note in MSC use with an NMP circuit.

Both vascular endothelial growth factor and IL-8 are key mediators in IRI. In their ex-vivo NMP porcine lung model which perfused human-umbilical cord derived MSCs, Mordant and associates, observed elevated VEGF concentrations in the lung perfusate as well as suppression of IL-8 [7Mordant P, Nakajima D, Kalaf R, et al. Mesenchymal stem cell treatment is associated with decreased perfusate concentration of interleukin-8 during ex vivo perfusion of donor lungs after 18-hour preservation. J Heart Lung Transplant 2016; 35(10): 1245-54.
[http://dx.doi.org/10.1016/j.healun.2016.04.017] ]. It may be possible that with the use of both a ROS species and lymphocyte scavenger within the circuit, the pro-inflammatory induction of MSCs theorised by Caseraghi’s group is potentially overcome, therefore permitting favourable cellular expression of VEGF. Mordants work supports the use of ex-vivo NMP to pre condition the allograft for MSC acceptance by creating an environment for beneficial cellular MSC expression. The groups’ machine, however, is still at its infancy.

Delivery of MSCs in Mordants’ group was either endotracheally or intra-arterially in 18 animals [7Mordant P, Nakajima D, Kalaf R, et al. Mesenchymal stem cell treatment is associated with decreased perfusate concentration of interleukin-8 during ex vivo perfusion of donor lungs after 18-hour preservation. J Heart Lung Transplant 2016; 35(10): 1245-54.
[http://dx.doi.org/10.1016/j.healun.2016.04.017] ]. After a cold storage period of 18 hours, ex-vivo lung perfusion (EVLP) was sustained for 12 hours with administration of human umbilical cord MSCs after one hour. One of the main observations of the model was the retention of the cells during the first few minutes after retention. Measurement of CD73⁺ cells as biomarkers of MSCs concentration, revealed high perfusate retention (83%) after the first 2 minutes followed by 100% depletion by 30 minutes. The converse elevated concentrations of CD73⁺ in the leucocyte scavenger of the machine highlighted a critical limitation. Intravascular administration albeit more effective than endobronchial, produce high losses through the scavenger mechanism. The resultant demonstration of human DNA in the lung parenchyma after 12 hours by the group using PCR, confirms the action of the MSCs. The aforementioned expression of IL-8 in the perfusate validated the function of the MSCs during this period. A clear limitation of the NMP model reported is the duration of the CS time along with the machine perfusion. Eighteen hours of cold storage followed by 12 hours of NMP would be considered extreme for solid organ use. In lung transplantation, current clinical practise is to ex vivo perfuse for upto four hours. Even though the authors’ have not commented, with further optimization, it may be possible that MSCs infusion of the circuit would enable longer perfusion and storage times in order to establish VEGF expression and in turn initiate IR repair mechanisms prior to implantation. It is not clear from this model or report alone, the dose effect relationship of the delivered human umbilcal cord MSCs on IR and inflammatory repair on immunomodulation. A potential target for renal allograft NMP would be to examine the expression of interleukin factors within the NMP circuit at time intervals as well and stratify this against outcome in an experimental large animal model.

Preclinical studies confirmed the safe use of MSC with promising results that demonstrated the immunomodulatory potential of MSC and their possible role in anti-rejection and immunosuppression maintenance protocols in organ transplantation. However, early clinical studies struggled to show this optimism. This was explained by various reasons including the in vitro microenvironment, type and donor age, Isolation techniques and site of isolation [27Kaundal U, Bagai U, Rakha A. Immunomodulatory plasticity of mesenchymal stem cells: A potential key to successful solid organ transplantation. J Transl Med 2018; 16(1): 31.
[http://dx.doi.org/10.1186/s12967-018-1403-0] ]. A surge of trials is underway tackling these challenges including proposals [27Kaundal U, Bagai U, Rakha A. Immunomodulatory plasticity of mesenchymal stem cells: A potential key to successful solid organ transplantation. J Transl Med 2018; 16(1): 31.
[http://dx.doi.org/10.1186/s12967-018-1403-0] ] utilising genetic modifications and targeted delivery systems with P-Selectin Glycoprotien Ligand-1 [28Liao W, Pham V, Liu L, et al. Mesenchymal stem cells engineered to express selectin ligands and IL-10 exert enhanced therapeutic efficacy in murine experimental autoimmune encephalomyelitis. Biomaterials 2016; 77: 87-97.
[http://dx.doi.org/10.1016/j.biomaterials.2015.11.005] ] or Etanercept [29Park N, Rim YA, Jung H, et al. Etanercept-synthesising mesenchymal stem cells efficiently ameliorate collagen-induced arthritis. Sci Rep 2017; 7: 39593.
[http://dx.doi.org/10.1038/srep39593] ]. We propose a novel technique in using MSC during ex-vivo normothermic kidney perfusion utilising the plasticity of MSC in organ optimisation and minimising the effect of reperfusion injury through their anti-apoptotic and anti-inflammatory functions.