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.
Highlights
As the outcome of kidney transplantation has improved, the demand for kidney transplantation has increased and the donor organ pool to date is too small to supply the current need for transplant organs
Isolation and Culture of Human and Porcine Mesenchymal stromal cells (MSC) and Endothelial Cells human MSC (hMSC) were isolated from subcutaneous adipose tissue from healthy human kidney donors (n = 5) that became available during kidney donation procedures after obtaining written informed consent as approved by the Medical Ethical Committee of the Erasmus University Medical Center Rotterdam (MEC2006-190). Porcine mesenchymal stromal cells (pMSC) were isolated from subcutaneous adipose tissue (n = 5) collected from male pigs, which were subjected to surgery for teaching purposes, as a waste product. hMSC and pMSC were isolated as described previously [25] and phenotypically characterized by the expression of CD29, CD44, CD90 and the absence of CD31 and CD45
Perfusion fluid had no negative impact on pMSC survival compared to culture medium except on thawed MSC after 30 min in perfusion (Figure 1A). hMSC were more resistant to suspension conditions than pMSC
Summary
As the outcome of kidney transplantation has improved, the demand for kidney transplantation has increased and the donor organ pool to date is too small to supply the current need for transplant organs. The shortage in available donor kidneys [1] has led to the use of expanded criteria donor organs, that includes kidneys from older donors or from donors with hypertension, suboptimal kidney function or death resulting from stroke [2]. This has resulted in a higher decline rate at time of offering and may lead to a poorer outcome of the transplantation [3]. Several techniques are being employed to improve the quality of expanded criteria kidneys and discarded kidneys to make them suitable for transplantation, including machine perfusion. NMP is postulated as a promising platform to reduce kidney damage and initiate regeneration prior to transplantation
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