Effects of Normothermic Machine Perfusion Conditions on Mesenchymal Stromal Cells

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Abstract

Ex-situ normothermic machine perfusion (NMP) of transplant kidneys allows assessment of kidney quality and targeted intervention to initiate repair processes prior to transplantation. Mesenchymal stromal cells (MSC) have been shown to possess the capacity to stimulate kidney repair. Therefore, the combination of NMP and MSC therapy offers potential to repair transplant kidneys. It is however unknown how NMP conditions affect MSC. In this study the effect of NMP perfusion fluid on survival, metabolism and function of thawed cryopreserved human (h)MSC and porcine (p)MSC in suspension conditions was studied. Suspension conditions reduced the viability of pMSC by 40% in both perfusion fluid and culture medium. Viability of hMSC was reduced by suspension conditions by 15% in perfusion fluid, whilst no differences were found in survival in culture medium. Under adherent conditions, survival of the cells was not affected by perfusion fluid. The perfusion fluid did not affect survival of fresh MSC in suspension compared to the control culture medium. The freeze-thawing process impaired the survival of hMSC; 95% survival of fresh hMSC compared to 70% survival of thawed hMSC. Moreover, thawed MSC showed increased levels of reactive oxygen species, which indicates elevated levels of oxidative stress, and reduced mitochondrial activity, which implies reduced metabolism. The adherence of pMSC and hMSC to endothelial cells was reduced after the thawing process, effect which was particularly profound in in the perfusion fluid. To summarize, we observed that conditions required for machine perfusion are influencing the behavior of MSC. The freeze-thawing process reduces survival and metabolism and increases oxidative stress, and diminishes their ability to adhere to endothelial cells. In addition, we found that hMSC and pMSC behaved differently, which has to be taken into consideration when translating results from animal experiments to clinical studies.

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Most cited references 31

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Renal transplantation after ex vivo normothermic perfusion: the first clinical study.

M L Nicholson, S A Hosgood (2013)

Ex vivo normothermic perfusion (EVNP) is a novel method of preservation that restores circulation and allows an organ to regain function prior to transplantation. The aim of this study was to assess the effects of EVNP in kidneys from marginal donors. Eighteen kidneys from extended criteria donors (ECD) underwent a period of EVNP immediately before transplantation. Kidneys were perfused with a plasma free red-cell based solution at a mean temperature of 34.6°C. The outcome of these kidneys was compared to a control group of 47 ECD kidneys that underwent static cold storage (CS). The mean donor age was 61 ± 1 years in the EVNP and 62 ± 6 years in the CS group (p = 0.520). EVNP kidneys were perfused for an average of 63 ± 16 min and all were transplanted successfully. The delayed graft function rate (DGF), defined as the requirement for dialysis within the first 7 days was 1/18 patients (5.6%) in the EVNP group versus 17/47 (36.2%) in the CS group (p = 0.014). There was no difference in graft or patient survival at 12 months (p = 0.510, 1.000). This first series of EVNP in renal transplantation demonstrates that this technique is both feasible and safe. Our preliminary data suggests that EVNP offers promise as a new technique of kidney preservation.

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Mesenchymal stem cells use IDO to regulate immunity in tumor microenvironment.

I Roberts, Liying Zhang, W Ling … (2014)

Mesenchymal stem cells (MSC) are present in most, if not all, tissues and are believed to contribute to tissue regeneration and the tissue immune microenvironment. Murine MSCs exert immunosuppressive effects through production of inducible nitric oxide synthase (iNOS), whereas human MSCs use indoleamine 2,3-dioxygenase (IDO). Thus, studies of MSC-mediated immunomodulation in mice may not be informative in the setting of human disease, although this critical difference has been mainly ignored. To address this issue, we established a novel humanized system to model human MSCs, using murine iNOS(-/-) MSCs that constitutively or inducibly express an ectopic human IDO gene. In this system, inducible IDO expression is driven by a mouse iNOS promoter that can be activated by inflammatory cytokine stimulation in a similar fashion as the human IDO promoter. These IDO-expressing humanized MSCs (MSC-IDO) were capable of suppressing T-lymphocyte proliferation in vitro. In melanoma and lymphoma tumor models, MSC-IDO promoted tumor growth in vivo, an effect that was reversed by the IDO inhibitor 1-methyl-tryptophan. We found that MSC-IDO dramatically reduced both tumor-infiltrating CD8(+) T cells and B cells. Our findings offer an important new line of evidence that interventional targeting of IDO activity could be used to restore tumor immunity in humans, by relieving IDO-mediated immune suppression of MSCs in the tumor microenvironment as well as in tumor cells themselves. ©2014 AACR

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Cryopreserved Mesenchymal Stromal Cells Are Susceptible to T-Cell Mediated Apoptosis Which Is Partly Rescued by IFNγ Licensing.

Raghavan Chinnadurai, Ian B. Copland, Marco A. S. Garcia … (2016)

We have previously demonstrated that cryopreservation and thawing lead to altered Mesenchymal stromal cells (MSC) functionalities. Here, we further analyzed MSC's fitness post freeze-thaw. We have observed that thawed MSC can suppress T-cell proliferation when separated from them by transwell membrane and the effect is lost in a MSC:T-cell coculture system. Unlike actively growing MSCs, thawed MSCs were lysed upon coculture with activated autologous Peripheral Blood Mononuclear Cells (PBMCs) and the lysing effect was further enhanced with allogeneic PBMCs. The use of DMSO-free cryoprotectants or substitution of Human Serum Albumin (HSA) with human platelet lysate in freezing media and use of autophagy or caspase inhibitors did not prevent thaw defects. We tested the hypothesis that IFNγ prelicensing before cryobanking can enhance MSC fitness post thaw. Post thawing, IFNγ licensed MSCs inhibit T cell proliferation as well as fresh MSCs and this effect can be blocked by 1-methyl Tryptophan, an Indoleamine 2,3-dioxygenase (IDO) inhibitor. In addition, IFNγ prelicensed thawed MSCs inhibit the degranulation of cytotoxic T cells while IFNγ unlicensed thawed MSCs failed to do so. However, IFNγ prelicensed thawed MSCs do not deploy lung tropism in vivo following intravenous injection as well as fresh MSCs suggesting that IFNγ prelicensing does not fully rescue thaw-induced lung homing defect. We identified reversible and irreversible cryoinjury mechanisms that result in susceptibility to host T-cell cytolysis and affect MSC's cell survival and tissue distribution. The susceptibility of MSC to negative effects of cryopreservation and the potential to mitigate the effects with IFNγ prelicensing may inform strategies to enhance the therapeutic efficacy of MSC in clinical use. Stem Cells 2016;34:2429-2442.

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Author and article information

Contributors

Jesus M. Sierra Parraga: URI : http://loop.frontiersin.org/people/628528/overview

Henri G. Leuvenink: URI : http://loop.frontiersin.org/people/241076/overview

Bjarne K. Møller: URI : http://loop.frontiersin.org/people/233304/overview

Carla C. Baan: URI : http://loop.frontiersin.org/people/51610/overview

Bente Jespersen: URI : http://loop.frontiersin.org/people/558982/overview

Martin J. Hoogduijn: URI : http://loop.frontiersin.org/people/29382/overview

Journal

Journal ID (nlm-ta): Front Immunol

Journal ID (iso-abbrev): Front Immunol

Journal ID (publisher-id): Front. Immunol.

Title: Frontiers in Immunology

Publisher: Frontiers Media S.A.

ISSN (Electronic): 1664-3224

Publication date (Electronic): 10 April 2019

Publication date Collection: 2019

Volume: 10

Electronic Location Identifier: 765

Affiliations

[1] ¹Department of Internal Medicine, Erasmus Medical Center, University Medical Center Rotterdam , Rotterdam, Netherlands

[2] ²Nuffield Department of Surgical Sciences and Oxford Biomedical Research Centre, University of Oxford , Oxford, United Kingdom

[3] ³Department of Clinical Immunology, Aarhus University Hospital , Aarhus, Denmark

[4] ⁴Department of Renal Medicine, Aarhus University Hospital , Aarhus, Denmark

[5] ⁵Department of Surgery – Organ Donation and Transplantation, University Medical Center Groningen , Groningen, Netherlands

Author notes

Edited by: Michael Uhlin, Karolinska Institute (KI), Sweden

Reviewed by: Marcella Franquesa, Germans Trias i Pujol Health Science Research Institute (IGTP), Spain; Federica Casiraghi, Istituto Di Ricerche Farmacologiche Mario Negri, Italy

*Correspondence: Jesus M. Sierra Parraga j.sierraparraga@ 123456erasmusmc.nl

Martin J. Hoogduijn m.hoogduijn@ 123456erasmusmc.nl

This article was submitted to Alloimmunity and Transplantation, a section of the journal Frontiers in Immunology

Article

DOI: 10.3389/fimmu.2019.00765

PMC ID: 6469476

PubMed ID: 31024574

SO-VID: 6e1a5ff4-d67e-4c20-8854-4b4bdd519a31

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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

History

Date received : 25 January 2019

Date accepted : 22 March 2019

Page count

Figures: 8, Tables: 1, Equations: 0, References: 46, Pages: 11, Words: 6998

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Effects of Normothermic Machine Perfusion Conditions on Mesenchymal Stromal Cells

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Most cited references 31

Renal transplantation after ex vivo normothermic perfusion: the first clinical study.

Mesenchymal stem cells use IDO to regulate immunity in tumor microenvironment.

Cryopreserved Mesenchymal Stromal Cells Are Susceptible to T-Cell Mediated Apoptosis Which Is Partly Rescued by IFNγ Licensing.

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Cited by 13

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