The Development Of A New Organ Preservation Solution And It's Effects On Rat Lung Hypothermic Preservation

Background: Organ preservation solution is closely related to ischemia-reperfusion injury and maintenance of organ viability.Currently,Perfadex is an effective and the most widely used lung preservation solution.However,the total lung preservation time is limited(less than 12 hours),and the rate of primary graft failure(PGD)after transplantation is 10-20%.In addition,its high price limits its wide application in the world.Therefore,it is imperative to develop a new economical and effective lung preservation solution.Method: The lung epithelial cells(BEAS-2B)or human microvescular pulmonary endothelial cells(HPMEC)were cultured to 80-90% confluency,the cell culture medium was removed,and the under testing preservation solutions were added to the cells respectively,such as Perfadex,PBS,0.9% sodium chloride,Steen solution,DMEM,DMEM + 10% FBS(D10),etc.Then the cells were put into a chamber at 4 ° C which was filled with 50% O2(including or excluding 5% CO2)to simulate the process of cold ischemia.18 hours or after 42 hours,the cells were switched back to the conventional cell culture medium environment at 37 ° C to simulate the process of reperfusion for 4 hours.Cell activity was then assessed using the MTS assay.We developed a new organ preservation solution JT-1,and tested it with a rat lung cold ischemia model,the rat lungs were preserved in different preservation solutions at 4? for 24 h,then lung tissue was collected and analyzed.Results: Cell culture media contains well-defined essential amino acids,vitamins and other nutrients.Interestingly,in comparison with Perfadex,DMEM,D10 and Steen solution significantly increased cell death,even significantly worse than PBS and 0.9% Na Cl,when used during preservation period.We suspected that the detrimental effects might be due to the bicarbonate buffer in these solutions.We then mixed 50% O2 with 5% CO2 during the preservation;CO2 can help to maintain the normal p H in media containing bicarbonate buffer.Indeed,cell viability were dramatically improved in DMEM and D10,and significantly better than that in Perfadex and PBS,in which cell viability was not affected by the 5% CO2.Cells preserved in 0.9% Na Cl showed increased cell death in the presence of 5% CO2.We then switched bicarbonate buffer with 40 m M HEPES in DEME,D10 and Steen solution,which significantly increased cell viability(p<0.001).We tested the new organ preservation solution JT-1 on a rat lung cold ischemia model,the results showed that the new organ preservation solution JT-1 had much better effects on protecting the rat lungs from ischemic injury during cold storage.Conclusion: 1?As a convenient screening tool,cell culture model allows testing multiple variables simultaneously.It also allows to test hypothesis and underlying mechanisms for lung preservation.2?The phosphate buffer system in Perfadex is essential for cyto-protection.3?The bicarbonate buffer in DMEM prevents its use as a preservation solution.4?However,the superior results when 5% CO2 or HEPES was added to DEME during preservation suggest that certain nutrients in DMEM should be considered to add to preservation solutions.5?The new organ preservation solution JT-1 had much better effects on protecting the rat lungs from ischemic injury during cold storage that Perfadex.