How to Freeze Extra Primary Cell Products

The below information is for customers who have extra fresh cell products or have fresh cells that are not needed immediately for processing. Work through the following steps quickly and without interruption. Please note once Cryopreservation Media is added the process cannot be stopped and should be followed through to completion. Freezing cells risks poorer viability and poorer cell recovery after thaw.

Reagent and Materials Required

• Cryopreservation Media (80% FBS, 20% DMSO)

• Isocove's Modified Dulbecco's Medium (IMDM)

• Ice

Equipment Required

• Refrigerated centrifuge

• Biosafety hood (Class II)

• Compound microscope

• 2mL Cryogenic vials

• Freezing rack

• 1 to 10 mL pipets

• Conical centrifuge tubes

• Isopropanol graded freezing containers

• -20oC freezer

• -80oC freezer

Procedure

1. Prepare Cryopreservation Media up to 2 days in advance and store at 4oC.

2. Label cryogenic vials with the appropriate vial and donor information.

3. Place ice at the bottom of a freezing rack, place vials in the freezing rack, store in a -20oC freezer for at least 10 minutes until ready for use.

4. Count the cells with preferred primary cell counting method.

5. Centrifuge cells at 200 x g for 5 minutes at 4oC and remove supernatant from cell pellet.

6. Based on the desired concentration of cells/vial, calculate the amount of IMDM required to resuspend cells using the formulas:

A) # cells/desired concentration of cells per vial = # vials needed

B) 0.9mL/vial X # vials = # mL of Cryopreservation Media required.

Resuspend the cell pellet in this volume of cold (4oC) IMDM.

7. Mix the suspension well by pipetting up and down. Incubate cells on ice for approximately 5 to 10 minutes.

8. In a small bowl, add ice and water to make a homogenous ice-water bath.

9. Place the cell suspension tube in the ice-water bath. Add the Cryopreservation Media to the cell suspension at a rate of 3 to 5 seconds per drop. Shake the tube constantly to ensure even mixing.

10. Remove freezing rack from freezer and place in the hood. Mix the cells well by pipetting and aliquot 1.8mL of cells to each cryogenic vial.

11. Freeze cells at a controlled rate of 1oC/min by placing vials in an alcohol-graded freezing container(s) and incubating at -80oC for a minimum of 4 hours to a maximum of overnight.

12. Transfer vials to vapor phase of liquid-nitrogen storage.

Cells stored in the vapor phase can be stored long-term up to several years. When commencing downstream applications on the cells,follow the protocol on how to thaw cells by referring to the How to Thaw Frozen Cells written guide.