Maintaining PBMC Cultures
ReagentVendorCatalog Number
Corning™ Costar™ Ultra-Low Attachment Microplates Fisher Scientific 07-200-601
QBSF-60 Serum Free Media Fisher Scientific NC0823508
rhSCF R&D Systems 255SC010CF
rhEPO R&D Systems 287TC500
rhIL-3 R&D Systems 203IL010CF
rhIGF-1 R&D Systems 291G1200
Dexamethasone Sigma D88931MG
L-Ascorbic Acid Sigma A454425G
2-mercaptoethanol Thermo Fisher / Life Technologies 21985023
Required Equipment / Consumables:
  • Biosafety Cabinet
  • CO2, O2, humidified, water jacketed incubator
  • Sterile, glass, borosilicate pipets (Fisherbrand)
  • Cellometer K2 Cell Counter (Nexcelom)
  • Prepare Media:
    PBMC Media (Make fresh prior to each media change):
    • QBSF-60 media – 10 mL
    • 50 ug/mL L-Ascorbic Acid
    • 2 U/mL EPO
    • 50 ng/mL SCF
    • 10 ng/mL IL-3
    • 40 ng/mL IGF-1
    • 1uM Dexamethasone
    Freezing Media:
    • 60% FBS
    • 20% QBSF-60 media
    • 20% DMSO
    PBMC Thawing:
    1. Remove PBMC vial(s) from liquid nitrogen tank and move it rapidly to a 37°C water bath.
    2. Count viable cells using AOPI staining and Cellometer
    3. Spin at 300xg for 10 minutes.
    4. Resuspend pellet in 2.5 mL of PBMC media
    5. Transfer to a ultra low attachment surface 6-well plate
    6. Incubate 37°C
    Expanding PBMC:
    1. Provide new PBMC media every 2-3 days
    2. Transfer cells to a 15 mL conical tube
    3. Add respective volume of QBSF-60 to dish and gently scrape dish with cell scrapper to collect loosely adherent cells and transfer to 15 mL conical
    4. Centrifuge cells 300 x g for 10 minutes
    5. Resuspend pellet in 2.5 mL of PBMC media
    6. Plate on 1 well of ultra low attachment surface 6-well plates
    7. Incubate 37°C
    hESC Freezing:
    1. Pre-label cryotubes and place in the freezer prior to freezing cells
    2. Transfer cells to a 15 mL conical tube
    3. Add respective volume of QBSF-60 to dish and gently scrape dish with cell scrapper to collect loosely adherent cells and transfer to 15 mL conical
    4. Count viable cells using AOPI staining and Cellometer
    5. Centrifuge cells 300 x g for 10 minutes
    6. Aspirate, and gently resuspend colonies in PBMC media, 500 uL per cryotube intended on freezing
    7. Add of 2x freezing media dropwise to colonies in PBMC media slowly, 500 uL per cryotube intended on freezing. Gently invert to mix, then dispense into the appropriate number of tubes
    8. Place in freezing container and store at -80°C overnight. The next day, transfer to liquid nitrogen. DO NOT LEAVE AT -80°C FOR EXTENDED PERIODS OF TIME