TITLE: Thawing Cryopreserved Human Islets
AUTHORS: James Lyon, Aliya Spigelman, Jocelyn E Manning Fox, Patrick Macdonald

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol details the thawing of human islets following their cryopreservation, as performed by the Alberta Diabetes Institute IsletCore. The crypreservation method is detailed in </span><a href="https://www.protocols.io/view/human-islet-cryopreservation-spcediw" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Human Islet Cryopreservation</span></a><span> protocol. </span></div><div class = "text-block">Human islets cryopreserved and thawed using these methods 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]
?. [M199 solution]
ABCDE1M199 (10L)2ReagentConcentrationMass/VolumeSupplierCatalogue #3 M199 powder9.41g/L
 1 bottle
 Mediatech-Corning90050PB 34 NaHCO326 mM
 22.0g
 Fisher ScientificS233-5005 HEPES10 mM
 23.83g
 Fisher ScientificBP310-16 Penicillin/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 into 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 stir into solution.Stir the solution for   Store the prepared solution overnight at   to allow all powders to go into the solution Stir the solution for   Calibrate the pH meter using the pH control buffersAdjust the pH level of the M199 solution to   using the NaOH and/or HCl.Bring to the volume of 10L with the appropriate amount of Milli-Q water (18mΩ).Stir the solution for Store the prepared solution overnight at  to allow all powders to go into the solution Stir the solution for Calibrate the pH meter using the pH control buffersAdjust the pH level of the M199 solution to   using the NaOH and/or HCl.Bring to the volume of 10L with the appropriate amount of Milli-Q water (18mΩ).Adjust the pH level of the M199 solution to  using the NaOH and/or HCl.Filter into 1L bottles with a 0.22µm nitrocellulose filter.
ABCDE1M199 (10L)2ReagentConcentrationMass/VolumeSupplierCatalogue #3 M199 powder9.41g/L
 1 bottle
 Mediatech-Corning90050PB 34 NaHCO326 mM
 22.0g
 Fisher ScientificS233-5005 HEPES10 mM
 23.83g
 Fisher ScientificBP310-16 Penicillin/Streptomycin20,000 U/ml penicillin and 20,000 μg/ml streptomycin50ml
 Lonza09-757F
9 L
4 °C
4 °C
?. [Sucrose Solution]
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 .
56 °C
56 °C
-20 °C
?. [Sucrose Solution]
Prepare 0.75M sucrose in freeze M199 and place on ice.Weigh  sucrose  and transfer to a 50 ml beaker.Add  of freeze M199 (as above) and mix until in solutionTransfer sucrose solution to a 50 ml volumetric flask, add freeze M199 to bring total volume to 50 ml.Filter sterilize with a 0.22 um cellulose acetate filter.
12.836 g
30 mL
?. [Human Islet Culture media]
ABCDEF1ReagentCMRL 1066Bovine serum albumin 30%Insulin-Transferrin-Selenium (100x)Gibco® GlutaMAX Supplement Penicillin-Streptomycin Mixture2Volume500 ml 8.5 ml5 ml10 ml2.5 mlPrepare Human Islet Culture media as described in the table above.  Filter sterilize using a Stericup Sterile Vacuum Filtration System.
ABCDEF1ReagentCMRL 1066Bovine serum albumin 30%Insulin-Transferrin-Selenium (100x)Gibco® GlutaMAX Supplement Penicillin-Streptomycin Mixture2Volume500 ml 8.5 ml5 ml10 ml2.5 ml
?. [Thawing protocol]
Remove tubes of cryopreserved human islets from Liquid Nitrogen dewar and place into portable bath containing liquid nitrogen.
?. [Thawing protocol]
Immerse and agitate tubes in  water bath until almost thawed (WARNING: do not over thaw, remove the tube from the water bath when a small piece of ice is still present).
37 °C
?. [Thawing protocol]
Transfer to  ice slush
0 °C
?. [Thawing protocol]
Centrifuge tubes at
Centrifuge: 282 34, 1 min, 4 10
?. [Thawing protocol]
Remove supernatant
?. [Thawing protocol]
Add  of cold 0.75M sucrose. Lightly hand vortex to get pellet back into suspension.  Hold on  ice slush for .
1 mL
0 °C
?. [Thawing protocol]
Transfer the samples to   for .
22 Room temperature
?. [Thawing protocol]
Continue at . Add  of freeze M199 and lighty mix. Incubate for .
0 Room temperature
1 mL
?. [Thawing protocol]
Add  of freeze M199 and lighty mix. Incubate for . .
1 mL
0 Room temperature
?. [Thawing protocol]
Add  of freeze M199 and lighty mix. Incubate for . .
2 mL
0 Room temperature
?. [Thawing protocol]
Add  of freeze M199 and lighty mix. Incubate for .
4 mL
0 Room temperature
?. [Thawing protocol]
Centrifuge the sample tubes at
Centrifuge: 282 34, 1 min
?. [Thawing protocol]
Remove supernatant and add  of Human Islet Culture media and lightly hand vortex to allow the islets to go into suspension.   .
5 mL
0 Room temperature
?. [Thawing protocol]
Using Human Islet Culture media, culture islets at   5% CO2 or use for experiments. It is recommended to hand pick the islets to purity as soon as possible after thawing.
22 °C
?. [Sucrose Solution]
Freeze M199:Prepare Freeze M199 solution as follows:Add  heat inactivated FBS  (HyClone™ Fetal Bovine Serum (Canada), Characterized - Fisher cat# SH3039603) to  M199.Store at .  Warm to room temperature for use.
100 mL
900 mL
4 °C