With the help of this method, we want to describe a procedure which is suitable to cryopreserve PBMCs in such a way that after thawing, the bioenergetic parameters are unchanged. One of the major experimental challenges of cryopreservation is that the freezing and thawing process can damage PBMCs, which affects the following bioenergetic measurements. With our protocol, we try to show a way to preserve PBMCs reliably without damage to the cells so that the measured bioenergetic parameters are not influenced.
The possibility of preserving PBMCs allows samples to be collected from different centers and measured at one location. It also makes measurement more time-independent. Cryopreservation of PBMCs allow studies to be performed simultaneously at multiple sites where the cells are collected and preserved.
The collected samples can then be measured centrally. This streamlines the process and planning of studies. To begin, add eight milliliters of DPBS into a sterile 50-milliliter conical tube.
To this, add eight milliliters of blood and use a three-milliliter plastic Pasteur pipette to mix the contents well. Add 15 milliliters of lymphocyte isolation medium into another conical tube. Tilt the tube by 20 to 30 degrees.
Then, use a three-milliliter Pasteur pipette to apply the first layer of the blood DPBS mix gently over the isolation medium. Layer the remaining mixture carefully over the side wall of the tube. Slowly bring the tube into an upright position to layer the remaining blood onto the blood layer.
Centrifuge the tube at 1, 000 G for 10 minutes at room temperature in a swinging bucket with the brakes off. The blood/PBMC mix will separate into four distinct layers. With a Pasteur pipette, remove 2/3 of the plasma layer, then use a one-milliliter pipette to collect the PBMCs.
Transfer the PBMCs into a new 50-milliliter tube until the entire layer has been harvested. Then, make the volume up to 25 milliliters with DPBS to remove residual isolation medium and other residues. Centrifuge the tube at 100 G for 10 minutes at room temperature with the brakes on.
Then, remove the supernatant with a vacuum pump. Next, resuspend the pellet in one-milliliter DPBS and add more DPBS to make up the volume to 25 milliliters to wash and resuspend the pellet. Cool a freezing container to four degrees celsius.
Then, place fetal bovine serum on ice. Resuspend the cell pellet containing the isolated PBMCs in one milliliter of fetal bovine serum. Next, mix DMSO with cooled serum on ice at a ratio of 1:5.
Transfer the cell suspension to two-milliliter labeled cryo tubes using one tube per collection tube. With an automated cell counter, determine the cell count and viability. Use a one-milliliter pipette to drop one milliliter of the DMSO/FBS mixture into the tube.
Then, place the tubes in the pre-cooled freezing container. Place the container in a freezer at minus 80 degrees Celsius for 24 hours. The next day, remove the tubes from the freezer and store them in the gas phase of liquid nitrogen.
Cell counts and viability were consistent before and after cryopreservation, indicating successful isolation and preservation.
