Feb 24, 2023

Public workspaceHuman Islet Isolation Media Preparation V.3

DOI
dx.doi.org/10.17504/protocols.io.5jyl8nnpdl2w/v3
  • 1University of Alberta
James Lyon
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.5jyl8nnpdl2w/v3
Protocol Citation: James Lyon, Aliya F Spigelman, Jocelyn E Manning Fox, Patrick E Macdonald 2023. Human Islet Isolation Media Preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8nnpdl2w/v3Version created by James Lyon
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 24, 2023
Last Modified: February 24, 2023
Protocol Integer ID: 77583
Abstract
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
Materials
MATERIALS
ReagentHanks balanced salts powderCorningCatalog #55-022-PB
ReagentMinimum Essential Medium Eagle modifided (EMEM) powderCorningCatalog #90-009PB
ReagentMedium 199 powderCorningCatalog #90-050-PC
ReagentSodium BicarbonateFisher ScientificCatalog #S233
ReagentCalcium ChlorideMP BiomedicalsCatalog #153502
ReagentMagnesium SulfateAmrescoCatalog #0338
ReagentHEPES Fisher ScientificCatalog #BP310
ReagentPenicillin/streptomycinLonzaCatalog #09-757F
ReagentAlbumin Bovine Serum (30%)equitech bio, inc.Catalog #160928-0262
ReagentBetadine Solution (10%)Stevens companyCatalog #036-08617
ReagentNicotinamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #N0636
Lympolyte 1.1, Cell Separation Media 1.10 g/ml - (Cedarlane Catalogue #CL6255)
ReagentBelzer UW Cold Storage SolutionBridge to LifeCatalog #092614
ReagentCMRL 1066 mediumCorningCatalog #15-110-CV
ReagentGlutamax (100x)Gibco - Thermo FischerCatalog #35050-061
ReagentInsulin Transferrin Selenium (20x)CorningCatalog #25-800-CR
ReagentDimethyl SulfoxideFisher ScientificCatalog #D128
ReagentNylon capsule filter 0.22uMmaine manufacturingCatalog #1213757
ReagentDithizoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #43820
ReagentStericup Sterile Vacuum Filtration System. Fisher ScientificCatalog #S2GPU05RE
Reagent0.45um Syringe FilterFisher ScientificCatalog #09-740-116

HBSS (perfusion, rinse, priming solution), M199 (wash solution) and EMEM (dilution solution)
HBSS (perfusion, rinse, priming solution), M199 (wash solution) and EMEM (dilution solution)

ABCDE
Hank's Balanced Salt Solution - HBSS (perfusion, rinse, priming solutions) (10L)
ReagentConcentrationWeight/VolumeSupplierCatalogue #
HBSS Powder9.51 g/L1 bottleMediatech/Corning55-022-PB
CaCl2 (anhydrous)3.60 mM4.0gMP Biochemicals LLC15350290
MgSO4 (anhydrous)0.81 mM0.98gVWR Life Science97061-438
NaHCO34.2 mM3.5gFisher ScientificS233-500
HEPES10mM23.83gFisher ScientificBP310-1
Penicillin/Streptomycin100 U/ml penicillin 100 μg/ml streptomycin50mlLonza09-757F

ABCDE
Medium 199 - M199 (Wash media) (10L)
ReagentConcentrationWeight/volumeSupplierCatalogue #
M199 powder9.41g/L1 bottleMediatech/Corning90050PB
NaHCO326 mM22.0gFisher ScientificS233-500
HEPES10 mM23.83gFisher ScientificBP310-1
Penicillin/Streptomycin100 U/ml penicillin 100 μg/ml streptomycin50mlLonza09-757F
ABCDE
Eagle Modified Minimal Essential Medium - EMEM (Dilution Media) (10L)
ReagentConcentrationWeight/volumeSupplierCatalogue #
EMEM powder9.23g/L1 bottleMediatech/Corning90009BP
NaHCO326 mM22.0gFisher ScientificS233-500
HEPES10 mM23.83gFisher ScientificBP310-1
Penicillin/Streptomycin100 U/ml penicillin 100 μg/ml streptomycin50mlLonza09-757F
Prepare and pH the HBSS, M199 and EMEM solutions
Prepare and pH the HBSS, M199 and EMEM solutions
  1. Prepare the HBSS, M199, and EMEM solutions outlined in step 1 of the Stock Media Preparation table using the following directions:
  2. Dispense Amount9 L of Milli-Q (18mΩ) water in to the carboy
  3. Store overnight at Temperature4 °C to allow to come to temperature.
  4. Using the stirrer add the media powder to the water and allow to go into solution.
  5. Add the powdered supplements and Penicillin/Streptomycin to the appropriate media based on the above tables and allow to stir into solution.
  6. Stir the solution for Duration00:30:00
  7. Store the prepared solution overnight at Temperature4 °C to allow all powders to go into solution
  8. Stir the solution for Duration00:30:00
  9. Calibrate the pH meter using the pH control buffers
  10. Adjust the pH level to Ph7.4 using the NaOH and/or HCl solutions.
  11. Bring to volume with the appropriate amount of Milli-Q water.







Filter sterilize the HBSS, M199 and EMEM solutions
Filter sterilize the HBSS, M199 and EMEM solutions
  1. 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.
  2. Store the filtered media at Temperature4 °C until needed for supplementation prior to isolation.

Prepare, pH and filter sterilize the 1M Nicotinamide solution
Prepare, pH and filter sterilize the 1M Nicotinamide solution
ABCDE
Nicotinamide Solution - 1M (1L)
ReagentConcentrationWeight/volumeSupplierCatalogue #
Nicotinamide powder1 M122.12Sigma Aldrich Canada CoN0636
HEPES10 mM2.383gFisher ScientificBP310-1

  1. Dispense Amount0.9 L of Milli-Q water in to the carboy.
  2. Store overnight at Temperature4 °C to allow to come to room temperature.
  3. Using the stirrer add the reagent powder into the water based on the table (Step 4) and allow to stir into solution.
  4. Stir the solution for Duration00:30:00
  5. Calibrate the pH meter using the pH control buffers
  6. Adjust the pH level to Ph7.4 using the NaOH and/or HCl solutions
  7. Bring to volume (1L) with the appropriate amount of Milli-Q water.
  8. Filter sterilize using a bottle top filter (0.22μM) into a sterile 1L bottle.



Preparation and use of Dithizone stain in Islet Preparations.
Preparation and use of Dithizone stain in Islet Preparations.
Preparation of dithizone

  1. Weigh out Amount0.2 g of dithizone powder into a 50ml conical tube.
  2. Add Amount6 mL of DMSO and mix until the powder is in solution.
  3. Bring the resulting dithizone solution to 40ml total volume with HBSS and mix.
  4. Transfer the dithizone solution to a 60cc syringe with a 0.45µm nylon filter.
Use
  1. For every ml of islet suspension add an equal amount of the prepared dithizone solution must be added to the sample.
  2. For visualization of staining add another Amount2 mL of HBSS to dilute the stain and reduce the background colour.
  3. Alternately, Amount100 µL of islet suspension, Amount100 µL dithizone and Amount200 µL HBSS.



Preparation of working solutions to be done the day of isolation.
Preparation of working solutions to be done the day of isolation.
To prepare the working solution supplement each of the listed stock medias with the indicated supplements.
ABCDEFGH
Total volume per bottleBSA 30% HBSSEMEMM199Nicotinamide (1M)Betadine
(ml)(ml)(ml)(ml)(ml)(ml)(ml)
SOLUTION
M199 (aliquot into 12 x 100ml)1172160 (4.0%)----------1000 12-----
Dilution - make 3 bottles2020----------2000-----20-----
Priming solution1000-----1000--------------------
Wash 1 - split into 2x 550ml110595 (2.5%)----------100010-----
Wash 2 - make 5 bottles105545 (1.125%)----------100010-----
Cannulation500-----500--------------------
Perfusion Solution350-----350--------------------
Decon 1300-----250---------------50
Decon 2250-----250--------------------
Decon 3250-----250--------------------
Collection tubes for collection of tissue during the purification step.
ABCDEFG
Gradient Collection tubes
tube #123456
ml of wash 2 solution100150200200225225
tube #789101112
ml of wash 2 solution225225225225225150

Preparation of gradient solutions for purification
Preparation of gradient solutions for purification
ABCDE
Density Gradients
lympholyte 1.100g/mlUW sol'n. 1.042 g/mlVolume (ml)Final Density g/ml
High density 77641411.074
Low density52881401.064
Human Islet Culture media
Human Islet Culture media
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.


ABCDEF
CMRL 1066Bovine serum albumin 30%Insulin-Transferrin-Selenium (100x)Gibco® GlutaMAX Supplement Penicillin-Streptomycin Mixture
(ml)(ml)(ml)(ml)(ml)
Culture media5008.55102.5
CMRL 1066 - Corning Catalogue # 15110CV
Bovine Serum Albumin 30% v/v - Equitech Bio Inc. Catalogue # BAL62
Insulin-Transferrin-Selenium (100x) - Corning Catalogue # 25800CR
Gibco® GlutaMAX™ Supplement (100x) - ThermoFisher catalogue # 35050061
Penicillin-Streptomycin Mixture - Lonza Catalogue # 09-757F