TITLE: Human Islet Isolation Media Preparation
AUTHORS: James Lyon, Aliya F Spigelman, Jocelyn E Manning Fox, Patrick E Macdonald

[DESCRIPTION]
This protocol describes the preparation of various media formulations required for the isolation and culture of human islets, as performed by the Alberta Diabetes Institute IsletCore. http://www.bcell.org/adi-isletcore.html

[STEPS]
SECTION: HBSS (perfusion, rinse, priming solution), M199 (wash solution) and EMEM (dilution solution)
1. 
 

 
 
 
 
 
SECTION: Prepare and pH the HBSS,  M199 and EMEM solutions
2. Prepare the HBSS, M199, and EMEM solutions outlined in step 1 of the Stock Media Preparation table using the following directions:
Dispense 9 L of Milli-Q (18mΩ) water in to the carboy 
Store overnight at 4 °C to allow to come to temperature.
Using the stirrer add the media powder to the water and allow to go into solution.
Add the powdered supplements and Penicillin/Streptomycin to the appropriate media based on the above tables and allow to stir into solution.
Stir the solution for 30 min 
Store the prepared solution overnight at 4 °C to allow all powders to go into solution 
Stir the solution for 30 min 
Calibrate the pH meter using the pH control buffers
Adjust the pH level to pH 7.4 using the NaOH and/or HCl solutions.
Bring to volume with the appropriate amount of Milli-Q water.

 






SECTION: Filter sterilize the HBSS, M199 and EMEM solutions
3. 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 4 °C until needed for supplementation prior to isolation.

SECTION: Prepare, pH and filter sterilize the 1M Nicotinamide solution
4.  

SECTION: Prepare, pH and filter sterilize the 1M Nicotinamide solution
5. Dispense 0.9 L of Milli-Q water in to the carboy. 
Store overnight at 4 °C to allow to come to room temperature. 
Using the stirrer add the reagent powder into the water based on the table (Step 4) and allow to stir into solution.
Stir the solution for 30 min 
Calibrate the pH meter using the pH control buffers
Adjust the pH level to pH 7.4 using the NaOH and/or HCl solutions
Bring to volume (1L) with the appropriate amount of Milli-Q water. 
Filter sterilize using a bottle top filter (0.22μM) into a sterile 1L bottle.



SECTION: Preparation and use of Dithizone stain in Islet Preparations.
6. Preparation of dithizone

Weigh out 0.2 g of dithizone powder into a 50ml conical tube.
 Add 6 mL of DMSO and mix until the powder is in solution.
 Bring the resulting dithizone solution to 40ml total volume with HBSS and mix.
 Transfer the dithizone solution to a 60cc syringe with a 0.45µm nylon filter.
 
Use
For every ml of islet suspension add an equal amount of the prepared dithizone solution must be added to the sample. 
For visualization of staining add another 2 mL of HBSS to dilute the stain and reduce the background colour.
Alternately, 100 µL of islet suspension, 100 µL dithizone and 200 µL HBSS.



SECTION: Preparation of working solutions to be done the day of isolation.
7. To prepare the working solution supplement each of the listed stock medias with the indicated supplements.
             Media Total per bottle BSA 25%  HBSS EMEM M199 HBSS Perifusion HBSS (priming) Nicotinamide     (mL) (mL) (mL) (mL) (mL) (mL) (mL) (mL)   M199 (aliquot x12) 1172 160   (4.0%) ----- ----- 1000  ----- ----- 12   Dilution #1, #2, #3 2020   (x3) ----- ----- 2000   (x3) ----- ----- ----- 20   Priming solution 1000 ----- ----- ----- ----- ----- 1000 -----   Wash 1 - split into 2x 550ml 1105   (x1) 95   (2.5%) ----- ----- 1000 ----- ----- 10   Wash 2 1055   (x5) 45 (1.125%) ----- ----- 1000   (x5) ----- ----- 10   Cannulation 500 ----- 500 ----- ----- ----- ----- -----   Perfusion Solution 350 ----- ----- ----- ----- 350 ----- -----   Decon 1 250 ----- 200 Betadine   50mL ----- ----- ----- -----   Decon 2 250 ----- 250 ----- ----- ----- ----- -----   Decon 3 250 ----- 250 ----- ----- ----- ----- -----  
  
         Gradient   Collection tubes               tube # 1 2 3 4 5 6   mL of wash solution 100 150 200 200 225 225   tube # 7 8 9 10 11 12   mL of wash solution 225 225 225 225 225 150  
 
SECTION: Human Islet Culture media
9. Preparation of the human islet culture media

Prepare the culture media by supplementing CMRL by the following table.  Following these additions to the culture media, filter sterilize the resulting solution through a 0.22 µm  Stericup Sterile Vacuum Filtration System. 


 


SECTION: Preparation of gradient solutions for purification
8.  
            Density   Gradients                   Bicoll   1.100g/ml  UW   sol'n. 1.042 g/ml  Volume   (ml)  Final   Density g/ml    High   density   77  64  141  1.074    Low   density  52  88  140  1.064