TITLE: IsletCore Equipment Setup for Human Islet Isolation
AUTHORS: Patrick MacDonald, James Lyon, Jocelyn Manning Fox

[STEPS]
?. [Room setup]
Turn on both biosafety cabinets (BSC) 20 minutes prior to use.  Be sure to disinfect the BSC work surfaces with 70% ethanol.Turn on COBE 2991 chiller to 4°C.Turn on chilling recirculating bath set to 4ºC (at least 4 hours prior to isolation start)Turn on centrifuge and set to program 1 (282xg, 4°C, 2 min). Be sure the centrifuge is equipped with centrifuge buckets designed to acoomadate 250 mL centrifuge tubes.Turn on Biorep perfusion system.Place biomedical waste receptacles in lab (1 box and 1 pail).Fill ice tray and bucket with ice and place in refrigerator
?. [Setup Auto-isolator]
Turn on the auto-isolator by turning on the power swtich at the back of the unit. Once logged in Press the "Isolator Protocol Wizard".Allow the shaker arm to initiateClick on the Next icon to continue to the “load isolator protocol” page
?. [Documentation]
Fill out relevant information related to the isolation using the donor information sheet.Prepare the Isolation worksheet.
?. [Setup Auto-isolator]
Select the “standard” protocol that has will load the pre-programmed steps for the protocol. This protocol is user defined.  ABCDEFGH1Stage nameFlow rate (ml/min)Chamber Temp (oC)Cooler (oC)Inlet Valve (On/Off)Outlet Valve (On/Off)Motion Profile (Vf,Vs,Rf,Rs)21Prime200374OffOff32Pause004OffOff43Fill300374OffOff54Digest 1300374OffOff(140,110,140,80)65Digest 2150374OffOff(140,110,140,80)76Collection 130004OnOn(140,110,140,80)87Pause004OnOn98Collection 230004OnOn(140,110,140,80)109Stop004OffOffVf-Vertical frequencyVs-Vertical strokeRf-Rotational frequencyRs-Rotational stroke
?. [Setup Auto-isolator]
Setup the digest/collection tubing lines as illustrated in an applicable BSC utilizing aseptic technique throughout procedure:Unwrap sterilized Ricordi chamber, secure top chamber to bottom chamber with steel clampUnwrap sterilized stainless steel heating coil and assemble to inlet tubing set and glass rods.Unwrap sterilized stainless steel cooling coil and assemble to outlet tubing set and glass rodsConnect sterile thermocouple to Ricordi chamber temperature port. Plug thermocouple into thermocouple monitor.Connect a 5cc syringe to the sampling port on the outlet tubing set.Place glass digestion rods in priming solution until the first step in the isolation profileInstall the now assembled tubing sets/Ricordi Chamber to the inlet valve, peristaltic pump, heating interface, infrared temperature controller, pressure transducer, shaking arm, outlet valve, and the cooling bath filled with 70% Ethanol.   aInitiate the isolator by using HBSS/priming solution in step 1 on the protocol “priming”.Observe the system for any problems (leaks, temperature, etc).Allow the system circulate until just before the completion of the pancreas perfusion in the Islet Isolation protocol.Initiate the isolator by using HBSS/priming solution in step 1 on the protocol “priming”.Observe the system for any problems (leaks, temperature, etc).Allow the system circulate until just before the completion of the pancreas perfusion in the Islet Isolation protocol.
?. [Set up the surgical area in preparation for cannulation and perfusion.]
Setup the surgical/perfusion equipment in an applicable BSC utilizing aseptic technique throughout procedure:Unwrap outer wrapping of sterilized cutdown tray and fold the wrapping underneath the tray as a sterile barrier.Connect cold water “in” and “out” lines to ports on cutdown tray. Tighten with wrench. Unclamp circulating cold water lines and turn on cold water bath (set to operate at 3°C).Remove air inside cutdown tray by raising water outlet port to allow air to escape.Open surgical pack (kidney bowl, mosquito forceps (x6), tissue forceps - toothed and non toothed, drummond forceps, silicon nitride marble set (9 marbles), micro scissors, Metzenbaum scissors (x2), 8x11 stainless steel tray) and open all sterile instruments and disposables for use during the decontamination and perfusion.Open cutdown tray screensPlace decontamination pack (3 sterile glass 500ml beakers) and pancreas container inside BSC.Open wrap of decontamination pack and pour decontamination medias 1, 2, and 3 into three separate sterile beakers.Label a sterile 15 mL conical tube as “Biopsy” with Isolation number (R#).Remove wrap from sterilized perfusion tray inside BSC.Connect cold water “in” line to bottom port on left side of perfusion chamber, and “out” line to top port.Connect the line in and line out water lines to perfusion tray inlet and outlet ports.Open perfusion tubing set and attach it to the perfusion tray and perfusion apparatus. Block temperature probe port on perfusion tray with 3cc syringe.Pour 500 mL of HBSS solution onto cutdown tray.Block temperature probe port on perfusion tray with 3cc syringe.Pour 500 mL of HBSS solution onto cutdown tray.
?. [Set up tissue collection following the digest]
Place circulating conical tube chilling block into BSC and connect cold water “in” line to bottom port and “out” line to top port. Tighten with wrench.Turn on cold water bath (set to operate at 3°C). Remove air inside chilling block by raising water outlet port to allow air to escape.Place 12 tubes filled with 100ml of Dilution 1 media into the cold blockTransfer inlet rod into 2L bottle of Dilution 2 mediaTransfer outlet rod into first tube in cold block in preparation of tissue dilution following the digestion step of the isolation.
?. [Set up Yankhauer and tissue collection bottle]
Obtain bucket with ice and place in BSCPlace sterile 1L glass media bottle with 300ml Wash 1 solution on ice.Assemble suction apparatus as follows:Place suction stand in BSC.Connect one of the vacuum lines to the suction canister and the house vaccum source.Add a second vaccuum line to the inlet of the cannister and the other end through the opening of the side of the BSCSterily attach a Yankauer suction tip to the inlet vacuum line and clamp it vertically tip down on the stand.Open vacuum valve.
?. [Perfusion appartus protocol]
The perfusion appartus should be pre-programed using the following profile:  ABCDE1StepTimePressure SPPressure SPTemp SP2mm Hgmm HgTray3minChannel AChannel B4124050504522401001004632401751754