Effects of Normothermic Machine Perfusion Conditions on Mesenchymal Stromal Cells

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.

In silico detection of SARS-CoV-2 specific B-cell epitopes and validation in ELISA for serological diagnosis of COVID-19

“…Third, conformational B-cell epitopes were predicted with DiscoTope2 on molecular structures of SARS-CoV-2 proteins 8 . The unprecedented speed at which experimental SARS-CoV-2 structures have been solved and deposited in the PDB allowed us to predict epitopes on 13 proteins (Table S3 ) 9 – 12 .…”

“…Current in-silico predictions suffer from sensitivity issues and this will only improve with larger training sets, i.e. more experimental structures of antigen–antibody complexes 8 . Nonetheless, our combined predictions accurately ranked the spike RBD and N-term nucleoprotein as top choice candidates for specific SARS-CoV-2 diagnostics, which was subsequently confirmed by ELISA serological tests.…”

Mesenchymal Stromal Cell Therapeutic Delivery: Translational Challenges to Clinical Application

“…Gregorini et al demonstrated that delivering MSCs during hypothermic machine perfusion improved outcomes in a rat model of ischemic kidney injury (107). Recently, Sierra Parraga et al demonstrated that machine perfusion of MSCs supports their function and survival, although more work will be required to determine the optimal conditions for perfusion (108). Notably, these strategies may reduce the need for immunosuppression to prevent organ rejection (109).…”

Cell therapy during machine perfusion

“…The effect of kidney NMP perfusion fluid constituents on the viability of MSC has been investigated. This study stimulated thawed and fresh MSC with perfusate and demonstrated MSC behaviour can be adversely affected by the conditions of the perfusion fluid [30]. Data showed that thawed MSCs have reduced viability in perfusion fluid, with reduced adherence to endothelial cells when compared with fresh MSCs.…”