Introduction: Ex Vivo Lung Perfusion (EVLP) uses a circulating perfusate to restore organ metabolism at normothermia to allow for donor lung assessment and treatment prior to transplant. Supplementing the lung perfusate with nutritional additives such as total parental nutrition (TPN) or glutamine (GlutaMAX) has extended porcine EVLP. However, to further prolong EVLP duration, which is required for advanced donor lung repairs, high throughput evaluation of the role of specific nutrients is required. We worked to optimize nutrient supplementation in the perfusate to address metabolic demands and maintain basic cell function of human endothelial cells in culture.

Methods: Mathematical models were developed to estimate nutrient and metabolic waste accumulation in the perfusate during EVLP. TPN, Intralipid and glutamine additives were assessed in different combinations based on a dose response curve in our high-throughput screening pipeline. Important cellular characteristics such as confluence, apoptosis, and migration, were evaluated in human pulmonary microvascular endothelial cells (HPMECs) and monitored on the Incucyte SX5 system. In total, 21 experimental perfusates were studied, with the current clinically used Steen solution serving as a control.

Results: HPMECs exposed to perfusates supplemented with TPN showed improved cell confluence and migration, and decreased apoptosis only up to 24h relative to the control (p<0.05). However, cells cultured in perfusates containing glutamine at 4 mM showed improved results in all three parameters at 24h and 48h (p<0.05). Intralipid supplementation alone did not differ from control. The combination of TPN, glutamine and Intralipid was similar to glutamine alone.

Conclusion: The addition of TPN and Intralipid to control EVLP perfusate showed benefits only until 24h, however the addition of glutamine demonstrated significant improvement in human endothelial cell function until 48h. This suggests that in addition to basic supplementation with TPN and Intralipid, further improvement in cell function can be achieved with the addition of glutamine. This cell culture model provides an important screening capability to develop enhancements of ex vivo lung perfusion solutions to better support donor lungs ex vivo.