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First Report on Ex Vivo Delivery of Paracrine Active Human Mesenchymal Stromal Cells to Liver Grafts During Machine Perfusion

Verstegen, Monique M.A.1; Mezzanotte, Laura2; Ridwan, R. Yanto2; Wang, Kairong PhD1,3; de Haan, Jubi MD4; Schurink, Ivo J.1; Sierra Parraga, Jésus M. MSc5; Hoogduijn, Martin PhD5; Kessler, Benedikt M. PhD6; Huang, Honglei6; Hall, Sean R.R.7; Ijzermans, Jan N.M.1; Löwik, Clemens W.G.M.2; van der Laan, Luc J.W.1; de Jonge, Jeroen MD, PhD1

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doi: 10.1097/TP.0000000000002986

Machine perfusion is rapidly becoming the standard for graft preservation and provides new opportunities for organ salvage, reconditioning, and repair. Based on their immune-modulatory and regenerating properties,1 arguable mesenchymal stromal cells (MSCs) are the most promising cell therapy for graft repair on the pump, though proof of concept is still lacking.2 Here, we show the feasibility of delivering clinically relevant numbers of human MSCs (hMSCs; 5–10 × 106/kg) during 30-minute hypothermic oxygenated machine perfusion in porcine liver grafts (n = 8; Figure 1). Full methods are described in Supplemental Materials and Methods (SDC, To track the biodistribution of hMSC, cells were genetically labeled with click beetle red luciferase.3 As shown in Figure 2, bioluminescent imaging of both the anterior and posterior side of the liver showed a wide range and patchy distribution of hMSC which retain throughout the liver after 30 minutes of perfusion. No significant difference between arterial (n = 4) or venous infusion (n = 4) was observed. Histological and RNA-expression analysis confirmed the delivery of the hMSC throughout the liver grafts. About 104–105 hMSC were estimated to be still present per kilogram of liver graft. Importantly, the hMSC retained their paracrine activity after infusion. Using both Luminex and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, increasing levels of human-specific interleukin-6 and interleukin-8 released in porcine blood were shown during 4-hour normothermic perfusion. In conclusion, this is the first report showing an effective delivery of hMSC in a liver machine perfusion model. Evidence of paracrine activity of hMSC after delivery indicates regenerative and immune-modulatory effects during normothermic graft perfusion, though this needs further research in a transplantation model.

Graphical abstract of the procedure. The timeline indicates the details of each time point. Pig livers are procured after a warm ischemia period of 30 min and infused with 5 × 106–107 click beetle red luciferase (CBred2)-labeled human mesenchymal stromal cell (hMSC) either via the portal vein or hepatic artery. The distribution of hMSC is visualized by bioluminescence imaging (BLI) immediately (time point 0) after infusion using an IVIS Imaging System 200. The liver is then placed on a purpose-made perfusion device in which it is perfused with cold (10°C) oxygenated University of Wisconsin (UW)-organ preservation fluid. After 30 min of perfusion, the distribution of the hMSC is again imaged using BLI. After imaging, the livers were perfused for 4 h with their own, heparinized, full blood at normothermic temperature (37°C) by ex vivo machine perfusion after which biopsies were collected for further analysis. The BLI signal is quantified, presence of hMSC was assessed in biopsies taken from left and right periphery and center of the liver using human-specific antibodies, proteomic and genetic analysis. HOPE, hypothermic oxygenated machine perfusion; qPCR, quantitative polymerase chain reaction.
Effective delivery of paracrine active human mesenchymal stromal cells (hMSCs) during normothermic hepatic machine perfusion. Live imaging of the livers (n = 8) infused with click beetle red luciferase (CBred2)-transduced hMSC in the hepatic artery (n = 4; A, B, E, F) or portal vein (n = 4; C, D, G, H) using the IVIS Imaging System 200, directly after infusion (directly after infusion, A, B, C, D) and after 30 min of perfusion (E, F, G, H). Images are representative images showing bright luciferin-based luminescence at the posterior (A, C, E, G) and anterior side (B, D, F, H) of the liver, demonstrating whole organ coverage of the infused hMSC. The presence of hMSC was confirmed by immunohistochemical staining for copepod green fluorescent protein (copGFP) (I arrows; ×40 objective) at end of perfusion, whereas no staining was observed in liver biopsies before infusion (J). Representative pictures are shown. Human-specific B2M gene expression was determined by nested quantitative polymerase chain reaction (qPCR) to confirm the presence of hMSC in the pig liver at the genetic level (M). Shown are the raw cycle threshold (Ct) values of B2M expression of 3 pigs. B2M expression was measured in the pig samples that were perfused with hMSC, (pig 1–3) and in nonperfused pig livers (n = 3). Luminex-based analysis using human-specific cytokines showed that interleukin (IL)-6 (O) and IL-8 (P) was secreted in the liver perfusate in 2 of the 4 pigs that were infused via the hepatic artery (filled symbols). In pig livers that were not infused with hMSC, no IL-6 or IL-8 was detected (open symbols). Human-specific IL-6 and IL-8 protein release was confirmed in the same 2 pig livers by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis (data not shown). nd, not detectable.


1. Fouraschen SMG, Hall SRR, de Jonge J, et al. Christ B, Oerlecke J, Stock P. Support of hepatic regeneration by trophic factors from liver-derived mesenchymal stromal/stem cells. In: Animal Models for Stem Cell Therapy. Methods in Molecular Biology (Methods and Protocols). 2014. New York, NY: Springer Science+Business Media89–104
2. Van Raemdonck D, Neyrinck A, Rega F, et al. Machine perfusion in organ transplantation: a tool for ex-vivo graft conditioning with mesenchymal stem cells? Curr Opin Organ Transplant. 2013; 18124–33
3. Hall MP, Woodroofe CC, Wood MG, et al. Click beetle luciferase mutant and near infrared naphthyl-luciferins for improved bioluminescence imaging. Nat Commun. 2018; 91132

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