TITLE: Human Islet Isolation Enzyme Preparation Version 1.0
AUTHORS: James Lyon, Aliya Spigelman, Jocelyn E Manning Fox, Patrick Macdonald

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the preparation of enzymes used in the isolation of human research islets by the Alberta Diabetes Institute IsletCore (www.bcell.org/adi-isletcore). It is to be used in concert with the other islet isolation protocols published by IsletCore.</div><div class = "text-block">The ADI IsletCore program is described in full here:</div><div class = "text-block"><span>Lyon J, Manning Fox JE, Spigelman AF, Kim R, Smith N, O'Gorman D, Kin T,  Shapiro AMJ, Rajotte RV, MacDonald PE (2016) Research-focused isolation  of human islets from donors with and without diabetes at the Alberta  Diabetes Institute IsletCore. </span><a href="http://press.endocrine.org/doi/abs/10.1210/en.2015-1562" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Endocrinology, 157(2): 560-569</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. [Preparation of HBSS Perfusion Solution - 10L]
Hank's Balanced Salt Solution - HBSS (perfusion, rinse, priming solutions) (10L)
ReagentFinal ConcentrationWeight/VolumeSupplierCatalgue NumberHBSS Powder9.51 g/L1 bottleMediatech/Corning55-022-PBCaCl2 (anhydrous)3.60 mM4.0gMP Biochemicals LLC15350290
MgSO4 (anhydrous)0.81 mM0.98gVWR Life Science97061-438NaHCO34.2 mM3.5gFisher ScientificS233-500HEPES10mM23.83gFisher ScientificBP310-1Penicillin/Streptomycin100 U/ml penicillin 100 μg/ml streptomycin50mlLonza09-757FDispense  of Milli-Q (18mΩ) water into the carboy Store overnight at  to allow to come to temperature.Using the stirrer add the media powder to the water and allow it to go into solution.Add the powdered supplements and penicillin/streptomycin to the appropriate media based on the above table and stir to allow it to go 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 buffersAdjust the pH level to   using the NaOH and/or HCl solutions.Bring to volume with the appropriate amount of Milli-Q water.Sterile filter using the peristaltic pump, the tubing (silicon 25 gauge) and the capsule filter (Nylon Capsule filter 0.22µm membrane)  into the appropriate sterilized media bottle. 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 buffersAdjust the pH level to   using the NaOH and/or HCl solutions.Bring to volume with the appropriate amount of Milli-Q water.Sterile filter using the peristaltic pump, the tubing (silicon 25 gauge) and the capsule filter (Nylon Capsule filter 0.22µm membrane)  into the appropriate sterilized media bottle. Adjust the pH level to  using the NaOH and/or HCl solutions.Bring to volume with the appropriate amount of Milli-Q water.Sterile filter using the peristaltic pump, the tubing (silicon 25 gauge) and the capsule filter (Nylon Capsule filter 0.22µm membrane)  into the appropriate sterilized media bottle. Store the filtered media at  until needed for supplementation prior to isolation.
Hank's Balanced Salt Solution - HBSS (perfusion, rinse, priming solutions) (10L)
ReagentFinal ConcentrationWeight/VolumeSupplierCatalgue NumberHBSS Powder9.51 g/L1 bottleMediatech/Corning55-022-PBCaCl2 (anhydrous)3.60 mM4.0gMP Biochemicals LLC15350290
MgSO4 (anhydrous)0.81 mM0.98gVWR Life Science97061-438NaHCO34.2 mM3.5gFisher ScientificS233-500HEPES10mM23.83gFisher ScientificBP310-1Penicillin/Streptomycin100 U/ml penicillin 100 μg/ml streptomycin50mlLonza09-757F
9 L
4 °C
4 °C
4 °C
?. [Reconstitution of Collagenase]
Obtain one vial collagenase powder from  storage and place it on ice.Under sterile conditions add  of perfusion solution to the vial of collagenase powder.Do not mix but allow the lyophilized cake of collagenase to settle into the solution for , gently swirling the bottle every 10 minutes to be sure to allow any residual collagenase that might be adhered to the glass to go into solution.After 30 min sterilely transfer the resulting collagenase concentrate to  of perfusion solution in a sterile 500 ml media bottle (on-ice)Rinse the original collagenase bottle with  of perfusion solution and transfer the rinse to the media bottle.Bring the total volume of the now working collagenase solution to approximately  using perfusion solution.Continue to store on ice until Step 7 of the Isolation of Human Pancreatic Islets of Langerhans for Research protocol.
-20 °C
30 mL
200 mL
20 mL
350 mL
?. [Reconstitution of Neutral Protease]
Obtain a vial of non-specific protease powder from   storage and place it on ice.Under sterile conditions remove the rubber stopper from the vial add of perfusion solution.Continue to store the vial on ice until Step 7 of the Isolation of Human Pancreatic Islets of Langerhans for Research protocol.Once the weight of the pancreas has been determined (see Step 7 of the Isolation of Human Pancreatic Islets of Langerhans for Research), remove a volume of non-specific protease solution that will equal to 25,000 U of enzyme per gram of pancreas weight.  The exact number of enzymatic units in each vial is lot dependant but the amount the number of units is stated by the manufacturer on the provided certificate of analysis.  This will be stated as 25,000,000 U/vial +/- 200,000 U.  For example a 100 g pancreas will require the full 10 ml of the non-specific protease solution to achieve 25,000 U/g of pancreas.  A 50 g pancreas will require 5 ml of the non-specific protease solution to achieve 25,000 U/g of pancreas.  The amount of non-specific protease should be adjusted to 12,500 U/g of pancreas when the cold ischemia time is greater than 10 hrs.
-20 °C
10 mL
?. [Preparation and use of the Collagenase / Non-Specific protease Solution]
Store the resulting solution on ice until Step 7(5) of the Isolation of Human Pancreatic Islets of Langerhans for Research protocol.
?. [Preparation and use of DNase Solution]
Obtain one vial of DNase I Grade II from bovine pancreas from   storage and place it on ice.Under sterile conditions remove the rubber stopper from the vial add  of perfusion solution.Continue to store the vial on ice until Step 8(6) of the Isolation of Human Pancreatic Islets of Langerhans for Research protocol.
4 °C
4 mL