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Cryobiology73(2016)20-29 ContentslistsavailableatScienceDirect Cryobiology
journal homepage:w w w.elsevier.comパocate/ycryo
Astudyonicecrystalformationbehavioratintracelularfrezingof plantcelsusingahigh-spedcamera
TakakoNinagawaa，AkemiEguchib，YukioKawamuraC，TadashiKonishid， AkiraNarumia，山
aGraduateScholofMechanicalEnginering，Kanagaw日lns封印teofTechnology，1030Shimo-ogino，Atsugi，K日，nagaw，日243回 0292，Jロpan bToyoEngineringWorks，LTD.，1634，Shimotsuruma，yiロm日to，Kanagawa，242-0001，Japan CCryobiofrontierRese日rchCente，rn日印ltyofAgricul印 re，lwateUniversity，Ueda3-18-8，Moriok日，lw口te，020-8550，Japan
CrosMark
dDepartmentofMechanicalEngine巾 g，OitaN凶
ARTICLE INFO
Artic/ehistory: Received30November2015 Receivedinrevisedform 9June2016 Acepted20June2016 Availableonline21June2016
Keywords: High-spedcamera Icecrystal Intracelularfrezing Plant Plasmamembrane Supercoling
1. Introduction
Cryopreservation techniques are used in various fields， inc1uding food and medical engine ering. The most ap propriate cryopreservationtemperaturedependsonthepropertiesofthe objectbeingpreserved，andthepreservationperiodvarieswith temperature. Cryopreservation involves placing living materials suchasfood，biologicalorgans，andtisuesunderalowtempera- ture，thusreducingputrefactionbybacteriaandplantrespiration [25].However，sincelivingmaterialscontainwater，theyarealso frozen spontaneously through supercoling when placed at a temperature below the solidification point. Therefore，
ホ Corespondingauthor.
E-mai/ ad dres ses: s12910 01@仁co.kanagawa-it.ac.jp (T. Ninagawa)， ykawa@
Iwate-u.a仁、jp (Y. I<awamura)， konishi@oita-c.tac.jp (T. I<onish，)i narumi@me. kanagawa-i.tac伊 (A.Narumi).
htp:/dx.doi.org/l0.1016/j.仁ryobio.12016.06.03 0011-2240f@2016ElsevierInc.A1Irightsreserved.
onalCol/egeofTechnology，16Mala，Oi臼，870-0152，Japan
ABSTRACT
Intracelulari仁ecrystalformation(IF)causesseveralproblemstocryopreservation，anditisthekeyto developingimprovedcryopreservationtechniquesthatcanensurethelong-termpreservationofliving tisues.Therefore，theabilitytocap凶 reclearintracelularfrezingimagesisimportantforunder- standingboththeocurenceandtheI1Fbehavio.rTheauthorsdevelopedanewcryomicroscopicsystem thatwasequipedwithahigh-spedcameraforthisstudyandsucesfulyusedthistocaptureclearer imagesoftheIFprocesintheepidermaltisuesofstrawberygeranium(SaxifragastoloniferaCurtis) leaves.Thissystemwasthenusedtoexaminepaternsinthelocationandformationofintracelularice crystalsandtoevaluatethedegreofceldeformationbecauseoficecrystalsinsidethecelandthe growingrateandgrainsizeofintracelularicecrystalsatvariouscolingrates.Theresultsshowedthat anincreaseincolingrateinfluencedtheformationpaternofintracelularicecrystalsbuthadlesofan efectontheirlocation.Moreover，itreducedthedegreofsupercolingattheonsetofintracelular frezingandthedegreofceldeformation;thecharacteristicgrainsizeofintracelularicecrystalswas alsoreduced，butthegrowingrateofintracelularicecrystalswasincreased.Thus，thehigh-sped cameraimagescouldexposethesechangesinIFbehaviorswithanincreaseinthecolingrate，and thesearebelievedtohavebeencausedbyanincreaseinthede。greofsupercoling.
2016ElsevierInc.Alrightsreserved.
cryopreservationtechniquescanbec1asifiedintotwocategories dependingonwhetherthelivingmaterialsarefrozen，withfrozen preservation general ly being considered to allow an extended preservationperiod.
Thesuitabilityoffrozenpreservationdependsonthestateofthe livingmaterialsduringcryopreservation.Mazur[19，20]previously describedageneralrelationbetweenthecolingrateandsurvival rateoffrezingcels，whichapliestobothanimalandplantcels. Ataslowcolingrate，theinsideofacelbecomesdehydrated because of the diference in chemical potential between the extracelular ice crystals and the intracelular solution [16，19]. Althoughabsolutedehydrationislethaltoacel，partialdehydra- tionincreasesthesurvivalrateoffrezingcels.Asthecolingrate increases，intracelularfrezingocursasaresultofsupercoling folowingextracelularfrezing[19，23，]whichisalsolethaltocels [1，16，23，31，34].However，afurtherincreaseinthecolingratecan leadtovitrificationpreservation，whichsignificantlyincreasesthe survival rate of frezing cels [19，27]. At present， however，