Shreffler:Cryopreservation

Overview

Cryopreserving PBMC allows for phenotyping or functional studies to be preformed at a future time for convenience or efficiency, but cryopreservation adversely affects the survival and function of leukocyte populations differentially and the user should make themselves aware of the literature on this.

This protocol has been adapted from the URECA study of the Inner City Asthma Consortium

Materials

• Sterile 50 mL conical tubes. Fisher #14-432-22
• Sterile 15 mL conical tubes. Fisher #14-959-53A
• Nalgene Cryo Freezing Container filled with 2-propanol. Fisher #15350-50 – 80° C Freezer
• Sterile, graduated transfer pipets. Fisher # 13-711-20 1.8 mL sterile cryogenic vials. Fisher # 12-565-171N
• 5 mL snap top tube (BD Falcon 12 x 75 mm) for RAST sample. Fisher #14-959-11A (Falcon # 352063)
• Biological safety cabinet. Forma Scientific Model 1286 or comparable vendor
• Sterile Dulbecco’s Phosphate buffered saline (PBS), Ca++ and Mg++ free. VWR#45000-436
• RPMI – 1640 plus antibiotics (Expires 1 month after preparing)
• AIM V Media (w/HAS). Invitrogen # 12055-091 (Expires 1 month after opening)
• Freezing Medium A. Tissue culture tested, heat inactivated, filtered, Human AB serum
• Freezing Medium B. 20% DMSO in Freezing Medium A
• Sterile 100 μL tips. Fisher # 21-381-8a.
• Non-sterile tips. Fisher # 21-197-8g
• Sterile Pasteur pipet (9”) Pipet, 10-100 μL. Fisher # 05-402-88
• Turk’s solution. VWR Catalog: #RC885016 – 500mL, #RC885032 – 1L
• Microcentrifuge tubes. Fisher # 02-681-290
• Serological pipet, 25 mL. Fisher # 13-678-14b
• Blood collection and basic lab supplies. Gloves, tube racks, sharps containers, microscope, Kimwipes, ethanol squirt bottle, cell counter, calculator, pen, paper, beaker for decanting, pipet aid, balance, boxes to hold cryovials, timer

Procedure

This is a sterile procedure. Perform all steps in a hood (biological safety cabinet), observing universal precautions for blood specimens and all biosafety regulations. All reagents should be used at room temperature.

1. Take Freezing Media A and B out of the freezer. For each 10 x 106 cells, 0.5 mL of A and B will be needed. Allow time for the media to reach room temperature.
2. After isolating and counting PBMC as per your protocol, pellet cells to be preserved one additional time, remove supernatant and loosen pellet for resuspension in freezing media.
3. Calculate the volume of PBS containing 1% Freezing Media A to slowly add (drop by drop to the side of tube to minimize shearing forces) to the cells to yield a concentration of 20 x 10⁶ cells/mL. Use a sterile pipette to add PBS containing 1% Freezing Media A to the cells to adjust the concentration to 20 x 10⁶ cells/mL. Mix gently.
4. Add an equal amount of Freezing Media B drop by drop while turning the tube. This should take 2 minutes. The final cell concentration will now be 10 x 10⁶ cells/mL.
5. Once mixing is complete, prepare the PBMC aliquots which will be frozen. Pipette gently to minimize sheer forces!
6. Aliquot to cryovials (10-20 million per tube)
7. Place the cryovials in a room temperature Nalgene freezing container with 2-propanol. Place the container in the freezer (– 80 °C) for at least 12 hours. After 12 hrs, the cryovials can be transferred to liquid nitrogen for long-term storage.

Discussion

discuss this protocol

References

1. Shreffler WG, Visness CM, Burger M, Cruikshank WW, Lederman HM, de la Morena M, Grindle K, Calatroni A, Sampson HA, and Gern JE. Standardization and performance evaluation of mononuclear cell cytokine secretion assays in a multicenter study. BMC Immunol. 2006 Dec 12;7:29. DOI:10.1186/1471-2172-7-29 |

Contact

• Who has experience with this protocol?
• Email Wayne Shreffler through OpenWetWare