Protocol – Thawing and Revival of Cryopreserved Cells

Overview:

• Cryopreservation is an efficient way of preserving cell culture. Preserved cells can be revived whenever needed.
• Cryopreservation involves freezing of cells at an ultra-low temperature below -135°C (e.g., liquid or gas phase of liquid nitrogen or -150°C ultra-low freezers) in a medium which contains cryoprotectant (e.g., DMSO, glycerol, etc).
• Cryoprotectants protect cells from death by reducing cell damage due to the formation of ice crystals. However, they also have adverse effects on cells, therefore, need to be removed at the time of revival.
• The revival of cryopreserved cells involves the rapid thawing of frozen cells. Rapid thawing protects cells from death by reducing the damaging effect of ice crystals during the rehydration process.

This protocol describes the revival process of cryopreserved adherent cells. The revival process involves rapid thawing of cryopreserved cells followed by removal of freezing medium which contains cryoprotectant, in this case, DMSO. The freezing medium can be removed immediately just after thawing the cells by centrifugation. Alternatively, cells are allowed to adhere to the culture dish followed by washing and addition of fresh growth medium.

Requirements

Reagents and solutions
> Complete Growth medium
> PBS

Equipment and disposables
> Culture vessel (T25 flask/ 60 mm dish)
> Sterile conical tubes (15-mL or 50-mL)
> Benchtop centrifuge with 45° fixed-angle or swinging-bucket rotor (e.g., Eppendorf™ 5804 Series)
> Personal protective equipment (sterile gloves, Cryo-Gloves, lab coat, Full-face protective mask/visor)
> Laminar flow hood
> Pipette tips and pipetman
> Serological pipettes and Pipetboy
> Inverted phase-contrast microscope

Starting material
Frozen cryopreserved cells of interest

Prior to start: 
Set the water bath at 37°C
Keep the growth medium in the water bath for warming up

Objective:
Revival and thawing of cryopreserved cells

Protocol

Step 1: Carefully take out cryovial from the liquid nitrogen container/ cryogenic ultra low-temperature freezer (-150°C or -180°C).
> Check the location of the cell line to be revived in your record.
> Carefully take out cryovial from the liquid nitrogen container/cryogenic ultra low-temperature freezer (-150°C or -180°C).

Tips:
> Carefully check the label and match with the storage record to ensure that it is the correct cells you want to revive.
> We recommend to revive one cell line at a time if you are not an expert in cell culture work.

Precaution:
> Follow all safety instructions (wear Cryo gloves, face shield, lab coat, etc.) while handling liquid nitrogen containers.

Step 2: Quickly thaw the cells.
> Place the cryovial in a 37°C water bath and agitate gently until the contents of the cryogenic vial are thawed completely.

Note:
> Thawing will take 1 – 2 minutes.

Step 3: Dilute the contents of the cryogenic vial with a growth medium
> Wipe the top of the cryogenic vial with 70% alcohol and bring inside the laminar flow hood.
> Transfer the contents from the cryogenic vial to a 15-ml centrifuge tube containing a 4-5 ml growth medium.

Note:
> You can directly transfer cells from cryogenic vial to a T25/ 60 mm culture dish containing 4 ml culture medium if you do not want to remove freezing medium immediately by centrifugation. In this case, resuspend the cells by gentle pipetting 2 – 3 times and place the flask in the cell culture incubator (see step 5).

Step 4: Harvest cells by centrifugation to remove the freezing medium.
> Centrifuge the tube for 5 – 10 min to  harvest cells
> Discard the supernatant. Dislodge the pellet by flicking the tube with your finger and resuspend the cells in a 5 ml growth medium.
> Transfer suspended cells to a T25 flask.

Tip
> We recommend using a T25 flask or 60 mm culture dish for the plating of revived cells. Sometimes revival is associated with a high number of cell death. In this case, you will have very few live cells 

Step 5: Wash cells with PBS and add fresh growth medium when the most cells are attached to the culture dish ( 8h  – 24 h)
> Inspect cells under an inverted microscope after 8 to 12 h.
> If cells are well attached, remove and discard the old medium which can contain freezing medium and dead floating cells.
> Add 4 – 5 ml PBS, swirl the flask gently, and remove PBS.
> Add 5 ml fresh culture medium.
> Incubate cells in tissue culture incubator.

Now cells are revived. Inspect them daily and replenish medium and subculture as required and recommended.