TITLE: Human Islet Cryopreservation Version 2.0
AUTHORS: James Lyon, Aliya Spigelman, Jocelyn E Manning Fox, Patrick Macdonald

[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 20 years 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 href="http://dx.doi.org/10.1007/s00125-015-3598-4" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Diabetologia, </span></a><a href="http://dx.doi.org/10.1007/s00125-015-3598-4" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">58(7): 1503-1512</span></a><span>.</span></div></div>

[STEPS]
?. [Preparation of Solutions]
ABCDE1M199 (10L)ConcentrationWeight/VolumeSupplierCatalogue #2M199 powder9.41g/L
1 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
Lonza09-757FPrepare the M199 solution using the above Media Preparation table above:Dispense  of Milli-Q (18mΩ) water in to a carboy Store overnight at  to allow to come to temperature.Using a stirrer add the M199 media powder to the water and allow to mix into solution.Add the powdered supplements and Penicillin/Streptomycin to the appropriate media based on the above table  and allow to stir into solution.Stir the solution for  Store the prepared solution overnight at   to allow all powders to go into solution Stir the solution for   Calibrate the pH meter using the pH control buffersStir the solution forStore the prepared solution overnight at  to allow all powders to go into solution Stir the solution for Adjust the pH level of M199 solution to using the NaOH and/or HCl.Bring to volume of  with the appropriate amount of Milli-Q water (18mΩ).Filter into  bottles with a 0.22µm nitrocellulose filter.
ABCDE1M199 (10L)ConcentrationWeight/VolumeSupplierCatalogue #2M199 powder9.41g/L
1 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
9 L
4 °C
4 °C
10 L
1 L
?. [Pre-cool cryopreservation bath]
Pre-cool FTS bath and set temperature at  starting temp approximately  prior beginning the cryopreservation.
-7.4 °C
?. [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]
Aliquot islets into the cryopreservation tubes with a maximum of 25,000 IEQ per cryopreservation tube.
?. [Protocol]
Centrifuge all tubes . After centrifugation, remove the supernatant.
Centrifuge: 280 34, 1 min, 4 10
?. [Protocol]
At room temperature, add of freeze M199 to each tube and suspend the islets.
1 mL
22 °C
?. [Protocol]
At room temperature, add  2M DMSO and start timer.
500 µl
22 °C
?. [Protocol]
At 5 minutes add an additional  2M DMSO.
500 µl
22 °C
?. [Protocol]
At 30 minutes add  3M DMSO.
2 mL
22 °C
?. [Protocol]
At 45 minutes transfer all tubes to a rack in an ice bath.
[ice bath]
?. [Protocol]
At 55 minutes transfer all tubes to the pre-chilled FTS bath (-7.5°C)
[Seeding bath]
?. [Protocol]
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]
At 85 minutes begin the FTS bath rate cooling procedure at 2.5°C/min
[cool down by 2.5°C/min]
?. [Protocol]
Once the FTS bath reaches  all tubes can be transferred to liquid nitrogen
-40 °C
?. [Storage]
Once the cryopreservation protocol is complete and the tubes have been transferred to liquid nitrogen (-190°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]
?. [Preparation of Solutions]
Freeze M199:Prepare Freeze M199 solution as follows:Add  heat inactivated FBS  (HyClone™ Fetal Bovine Serum (Canada), Characterized - Fisher cat# SH3039603) to  M199 media.Store at .  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  .Once   is reached, place the tubes in the water-bath for  .After 30 minutes immediately remove the tubes from the water bath. Set water-bath temperature to maintain the product at .Once  is reached, place the tubes in the water-bath for .Store the tubes of heat inactivated FBS at.
100 mL
900 mL
4 °C
56 °C
56 °C
-20 °C
?. [Preparation of Solutions]
3M DMSO:Add Freeze M199 to  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 at  .  Warm to room temperature prior to use.Store at .
[DMSO]
4 °C
?. [Preparation of Solutions]
2M DMSO:Add Freeze M199 to   (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 at  .  Warm to room temperature prior to use.Store at .
[DMSO]
4 °C