TITLE: Human Islet Cryopreservation
AUTHORS: James Lyon, Patrick MacDonald, Jocelyn Manning Fox

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details the cryopreservation of human islets, as performed by the Alberta Diabetes Institute IsletCore. Human islets cryopreserved using this method have been found to retain viability and function after 20years of cryogenic biobanking.</div><div class = "text-block"><span>Manning Fox JE, Lyon J, Dai XQ, Wright RC, Hayward J, van de Bunt M, Kin  T, Shapiro AMJ, McCarthy MI, Gloyn AL, Ungrin MD, Lakey JR, Kneteman  NM, Warnock GL, Korbutt GS, Rajotte RV, MacDonald PE (2015) Human islet function following 20 years of cryogenic biobanking. </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Diabetologia, </span></a><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">58(7): 1503-1512</span></a><span>.</span></div></div>

[STEPS]
?. [Pre-cool cryopreservation bath]
Pre-cool FTS bath and set temperature at –7.4°C starting temp approximately 2 hours prior beginning the cryopreservation.
?. [Protocol - Step 1]
Aliquot islets into the cryopreservation tubes with a maxiumum of 25,000 IEQ per cryopreservation tube.
?. [Label the cryopreservation tubes]
Label  cryopreservation tubes using Brady™ FREEZERBONDZ™ Polyester Thermal Transfer Printer Labels (Fisher Scientific Cat #22-500521). Include the following information.Internal Identifier number (Rxxx)Number of islet equivalents (IEQ) per tubeCryoprotectant (DMSO)Cryopreservation dateTube number
?. [Protocol - Step 2]
?. [Preparation of Solutions]
ABCDEF1M199 (10L)SupplierSupplier Catalogue #Link2M199 powder9.41g/L
1 bottles (1x10L/bottle)
Mediatech-Corning90050PB 33NaHCO326 mM
22.0g
Fisher ScientificS233-5004Hepes10 mM
23.83g
Fisher ScientificBP310-15Penicillin/Streptomycin20,000 U/ml penicillin and 20,000 μg/ml streptomycin50ml
Lonza09-757FFreeze M199:Prepare Freeze M199 solution as follows:Add 100ml heat inactivated FBS  (HyClone™ Fetal Bovine Serum (Canada), Characterized - Fisher cat# SH3039603) to 900ml M199Store ar 4°C.  Warm to room temperature prior to use.Heat inactivation of FBSThaw serum and aliquot into labelled 50ml tubes. If serum was thawed in a refrigerator allow serum to come to room temperature prior to placing in water bath.Fill the water-bath with sufficient water so that the tubes may be submersed to the level of the serum.Set water-bath temperature to maintain the product at 56°C.Once 56°C is reached, place the tubes in the water-bath for 30 minutes.After 30 minutes immediately remove the tubes from the water bath. Store the tubes of heat inactivated FBS @ -20°C.2M DMSO:Add enough Freeze M199 to 7.8g of DMSO (Fisher Scientific - Cat# BP231)for a total volume of 50ml.Filter sterilize through a syringe filter (Nylon 0.2µm pore) into a sterile glass media bottleStore ar 4°C.  Warm to room temperature prior to use.3M DMSO:Add enough Freeze M199 to 11.7g of DMSO (Fisher Scientific - Cat# BP231)for a total volume of 50ml.Filter sterilize through a syringe filter (Nylon 0.2µm pore) into a sterile glass media bottle.Store ar 4°C.  Warm to room temperature prior to use.3M DMSO:Add enough Freeze M199 to 11.7g of DMSO (Fisher Scientific - Cat# BP231)for a total volume of 50ml.Filter sterilize through a syringe filter (Nylon 0.2µm pore) into a sterile glass media bottle.Store ar 4°C.  Warm to room temperature prior to use.
ABCDEF1M199 (10L)SupplierSupplier Catalogue #Link2M199 powder9.41g/L
1 bottles (1x10L/bottle)
Mediatech-Corning90050PB 33NaHCO326 mM
22.0g
Fisher ScientificS233-5004Hepes10 mM
23.83g
Fisher ScientificBP310-15Penicillin/Streptomycin20,000 U/ml penicillin and 20,000 μg/ml streptomycin50ml
Lonza09-757F
?. [Storage]
Once the cryopreservation protocol is complete and the tubes have been transferred to liquid nitrogen (-174.5°C), the tubes can be transferred to long term storage in a liquid nitrogen vapour phase.  This vapour phase can be achieved by using a Chart MVE 1500 Series LN2 storage tank or similar system.Human islets cryopreserved in this manner can be successfully thawed using the Thawing Cryopreserved Human Islets protocol.
[store in LN2 vapour phase]
?. [Protocol - Step 3]
Add 1ml of freeze M199 to each tube and suspend the islets. (RT)
[Freeze M199]
?. [Protocol - Step 4]
Add 500µl 2M DMSO and start timer.  (RT)
[2M DMSO]
?. [Protocol - Step 5]
At 5 minutes add an additional 500µl 2M DMSO -  (RT)
[2M DMSO]
?. [Protocol - Step 6]
At 30 minutes add 2ml 3M DMSO -  (RT)
[3M DMSO]
?. [Protocol - Step 7]
At 45 minutes transfer all tubes to a rack in an ice slush.(4°C)
[ice bath]
?. [Protocol - Step 9]
At 55 minutes transfer all tubes to the pre-chilled FTS bath (-7.5°C)
[Seeding bath]
?. [Protocol - Step 10]
At 65 minutes nucleate* all tubes to release the latent heat of fusion*Nucleation is necessary to remove the latent heat of fusion.  At this point the tissue suspension is "super-cooled".  This refers to the fact that the suspension is liquid despite being at -7.5°C. Nucleation is achieved by submersing each tube for 2 seconds in liquid nitrogen and then applying a mechanical force (firmly hit the tube against a solid object).  If nucleation is successful the solution will appear to be a "slush".  Immediately after nucleation return each tube back to the seeding bath.
[Nucleation]
?. [Protocol - Step 8]
At 85 minutes begin the FTS bath rate cooling procedure at -2.5°C/min
[cool down by 2.5°C/min]
?. [Protocol - Step 11]
Once the FTS bath reaches -40°C all tubes can be transferred to liquid nitrogen