Antifreeze Protein Prolongs the Life-Time of Insulinoma Cells during Hypothermic Preservation.docVIP

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Antifreeze Protein Prolongs the Life-Time of Insulinoma Cells during Hypothermic Preservation.doc

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Antifreeze Protein Prolongs the Life-Time of Insulinoma Cells during Hypothermic Preservation

AntifreezeProteinProlongstheLife-TimeofInsulinoma CellsduringHypothermicPreservation TatsuroKamijima1,MamiSakashita1,AiMiura1,YoshiyukiNishimiya1,SakaeTsuda1,2* 1BioproductionResearchInstitute,NationalInstituteofAdvancedIndustrialScienceandTechnology(AIST),Sapporo,Hokkaido,Japan,2GraduateSchoolofLifeScience, HokkaidoUniversity,Sapporo,Hokkaido,Japan Abstract It is sometimes desirable to preserve mammalian cells by hypothermia rather than freezing during short term transplantation. Here we found an ability of hypothermic (+4uC) preservation of fish antifreeze protein (AFP) against rat insulinoma cells denoted as RIN-5F. The preservation ability was compared between type I–III AFPs and antifreeze glycoprotein(AFGP),whichcouldberecentlymass-preparedbyadevelopedtechniqueutilizingthemusclehomogenates, butnotthebloodserum,ofcold-adaptedfishes.ForAFGP,whosemolecularweightisdistributedintherangefrom2.6to 34kDa,onlytheproteinslessthan10kDawereexamined.Theviabilityratewasevaluatedbycountingofthepreserved RIN-5F cells unstained with trypan blue. Significantly, either AFPI or AFPIII dissolved into Euro-Collins (EC) solution at a concentrationof10mg/mlcouldpreserveapproximately60%ofthecellsfor5daysat+4uC.The5-daypreservedRIN-5F cellsretainedtheabilitytosecreteinsulin.Only2%ofthecellswere,however,preservedfor5dayswithoutAFP.Confocal photomicroscopyexperimentsfurthershowedthesignificantbindingabilityofAFPtothecellsurface.Theseresultssuggest thatfishAFPenables5-dayqualitystorageoftheinsulinomacellscollectedfromadonorwithoutfreezing. Citation: Kamijima T, Sakashita M, Miura A, Nishimiya Y, Tsuda S (2013) Antifreeze Protein Prolongs the Life-Time of Insulinoma Cells during Hypothermic Preservation.PLoSONE8(9):e73643.doi:10.1371/journal.pone.0073643 Editor:MatsuoUemura,IwateUniversity,Japan ReceivedMay31,2013;AcceptedJuly19,2013;PublishedSeptember17,2013 Copyright: ? 2013 Kamijima etal. This is an open-access article distributed under the terms of the Creative Commons Attribution L