Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

PreparationofIsletsforTransplant(Tx)

1. Underaninvertedmicroscope,hand-pickisletsusingaP200pipetmanandstraightpipettipfromtheculturedisletsina100mmplate.
2. Countislets100atatimeandtransferintoeachmicrocentrifugetube(~500islets/tube/mouse).
3. Allowisletstosettletothebottomofthemicrocentrifugetubes.
4. DrawoneisletpelletintoaP200pipetman(setat130ul)usingastraightthin-wallpipettip.
5. Placeasiliconetubeadapteroverthesyringetip.InsertalengthofPE50tubingintothesiliconeadapter.
6. HangthepipetmantothesideofthehoodandtapethePE50tubingtothehoodwallhigherthantheisletsinthetip.Thiswillallowtheisletstosettleonlyinthetipofthepipette.
7. TransfertheisletsintothePE50tubingbyslowlydialingthepipetmanandmovingtheisletsintothePE50tubing,beingcarefulnottoexpeltheisletsfromtheend.
8. MakeakinkinthebeveledendofthePE50tubing.Whilemaintainingthekink,disconnectthesiliconeadaptertubingfromthepipetmanandsecurethekinkwiththesiliconeadapter.
9. Placetheislets,inthePE50tubing,kinkedsiliconeadaptortubingsidedown,intoa15mLconicalwithacut5mLpipetandtapethePE50tubingoverthesideofthe5mLpipettopreventcurlingofthePE50tubingwhilecentrifuging.
10. CentrifugethePE50/15mLconicalsto1000rpmandturnoffcentrifuge.(Neverpreparemorethan10isletpreparationsatonetime).
11. Placetubeonice.

PreparationofMouseforTransplant

1. AnesthetizethemicewithisofluoraneorwithKetamine/Xylexene(seepreparationprotocol).
2. Afteranesthetichastakenaffect,shavetheleftflankofthemouse.
3. Swabskinofmouse,center-out,withPovidoneIodineswabandthenwipeoffwithanETOHswab.
4. Locatetheleftkidney(justrightofspleen).Makeasmallincisionintheskin,exposingtheperitoneum.
5. Makeasmallincisionintheperitoneumexposingthekidney.Keepingtheincisionsmallwillhelpinkeepingthekidneyraisedandexposed.
6. Applyaslightpressuretobothsidesoftheincision,raiseorpopthekidneyoutofthemouse.
7. Keepthekidneymoistbyapplyingnormalsalinewithacottontippedswab.
8. Usingasyringe23or25gaugeneedle,makeasmallscratchontherightflankofthekidney,creatinganickinthekidneycapsule;nottoodeeportoolarge.

TransplantationofIslets

Note:WhilethemouseisbeingpreparedforTx,thesecondpersonshouldpreparetheHamiltonscrew-driveislettransplantsyringe.

1. SlowlyremovethesiliconeadaptortubingfromthePE50tubingwhilekeepingthekinkinthetubing.
2. PlacetheoppositeendofthePE50tubingintothesiliconeadaptortubingandplacethesiliconeadaptortubingontothepipettetipattachedtothe"screw-drive"glassHamiltonsyringe.SlowlyreleasethekinkinthePE50tubing,makingsurethattheislesdonotleakout.
3. AdvancetheisletsslowlytothetipofthePE50tubingusingthe"screw"mechanism,butkeepasmallairbubbleinthefrontoftheisletsinthePE50tubing.
4. Intothenickmadeinthekidney,carefullyslidethePE50tubingunderthecapsule,makingasmallpocket.Beverycarefulnottogougethekidneyorpuncturingthroughthecapsule.
5. Ithelpstokeeptheareaandcapsulemoistwithnormalsaline-Gentamycinsoakedcottontippedswab;adrycapsulewillteareasily.
6. Gentlymovethetubinginalldirections,creatinga"pocket"forthetransplantedisletstorest.Remembertoreapplyadditionalnormalsalinetokeeptheareamoist.
7. UnderthedirectionofthepersonwhohasopenedthemouseandwhohasplacedthePE50tubingunderthekidneycapsule,thesecondperson,whopreparedthemouseandHamiltontransplantsyringe,willslowlyadvanceisletsunderthecapsule,insidethe"pocket",behindasmallairbubbledeliveredbythePE50tubing,untilalltheisletsaretransplanted.
8. SlowlyremovethePE50tubing,drytheareawithadryswabandcarefullycauterizethenickwithlowheat.
9. Usingadrycottontippedswab,makesureallbleedinghasstopped.Oncebleedinghasstopped,re-moistenthekidneywithsterilesaline,andgentlyreplacethekidneyintotheperitoneumpriortoclosingthemousewithsutureandskinstaples.

10. Closing/RevivalofMouse

    1. Closetheperitoneumwitharunningstitchusing5-0silksuturesw/aC-619mmneedle.
    2. Usingforcepsdrawbothsideoftheskinincisiontogether.
    3. Stapletheskintogetherwith2or3staples.
    4. Cleantheskinofthemouseofanyblood,usingacottontippedswabandsaline.
    5. ImmediatelytreatthemousewithasubcutaneousinjectionofFlunixinandBuprenorphine.
    6. Placethemouseinacage,thatisplacedonaheatingpadorbelowaheatinglamp,untilthemouseisfullyactive.
    7. Removetheskinstaplesin2weeks.