Materials

• Cryopreservation medium. Options include:
  • CryoStor® CS10 (Catalog #07930), or
  • Dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS)
• Cryogenic vials (e.g. Catalog #38053)
• Isopropanol freezing container (e.g. Corning® CoolCell® (Catalog #200-0642), Mr. Frosty™ or a controlled-rate freezer)
• Pipettor (e.g. Corning® Lambda™ Plus Pipettor, Catalog #38060)
• Pipette tips (e.g. Corning® Filtered Pipette Tips, Catalog #38034)

Protocol

Option 1: Cryopreservation with CryoStor® CS10

1. Wipe down the outside of the CryoStor® CS10 container with 70% ethanol or isopropanol before opening.
2. Label cryogenic vials.
3. Ensure PBMCs are in a single-cell suspension. Centrifuge cells at 300 x g for 10 minutes to obtain a cell pellet.
4. Carefully remove the supernatant with a pipette, leaving a small amount of medium to ensure the cell pellet is not disturbed.
5. Resuspend the cell pellet by gently flicking the tube.
6. Add cold (2 - 8°C) CryoStor® CS10, mix thoroughly, and transfer the suspension to a cryovial.
   
   
   Note: It is recommended to freeze isolated PBMCs at a concentration of 0.5 - 10 x 10⁶ cells/mL. However, you should try freezing the cells at multiple concentrations to determine which concentration gives the desired viability, recovery, and functionality upon thaw. Higher cell concentrations for freezing will need to be validated and optimized for specific applications as the major risk is the loss of viable cells.
7. Incubate cells at 2 - 8°C for 10 minutes.
8. Cryopreserve cells using a standard slow rate-controlled cooling protocol (approximately -1°C/minute) on a controlled-rate freezer, or an isopropanol freezing container (e.g. Corning® CoolCell®, Mr. Frosty™ etc.). If using an isopropanol freezing container, place the cryogenic vials into the container, and place it in a -80°C freezer overnight.
9. For long-term storage, transfer the vials of frozen PBMCs from the freezer or freezing container to vapor phase liquid nitrogen (below -­135°C). Minimize exposure to room temperature by placing vials on dry ice during transfer to liquid nitrogen.
   
   
   Note: Long-term storage at -80°C is not recommended.

Option 2: Cryopreservation in 10% DMSO with 90% FBS

Before You Begin:

• Ensure all media is cold prior to starting this protocol.
• If there are safety concerns related to using FBS, a serum- and animal component-free cryopreservation medium such as CryoStor® CS10 should be used.

1. Prepare 20% DMSO in FBS. Keep on ice.
   
   
   Note: Do not put 100% DMSO on ice or it will form crystals. Use a glass pipette for DMSO.
2. Label cryogenic vials.
3. Ensure PBMCs are in a single-cell suspension. Centrifuge cells at 300 x g for 10 minutes to obtain a cell pellet.
4. Carefully remove the supernatant with a pipette, leaving a small amount of medium to ensure the cell pellet is not disturbed.
5. Resuspend PBMCs in cold FBS to a concentration of 1 - 20 x 10⁶ cells/mL. Keep on ice.
6. Mix cells gently with 20% DMSO in FBS at a ratio of 1:1. The final cell suspension will be in 10% DMSO and 90% FBS. The final cell concentration will be between 0.5 - 10 x 10⁶ cells/mL. Rapidly transfer 1 mL of cell suspension to each cryovial.
   
   
   Note: You should try freezing the cells at multiple concentrations to determine which concentration gives the desired viability, recovery, and functionality upon thaw.
7. Place cryogenic vials immediately into an isopropanol freezing container (e.g. Corning® CoolCell®, Mr. Frosty™ etc.). Place the container in a -80°C freezer overnight.
   
   
   Note: Do not let cells sit in cryopreservation medium at room temperature. Keep on ice and transfer rapidly.
8. For long-term storage, transfer the vials of frozen PBMCs to vapor phase liquid nitrogen (below -135°C). Minimize exposure to room temperature by placing vials on dry ice during transfer from the -80°C freezer to liquid nitrogen.
   
   
   Note: Long-term storage at -80°C is not recommended.