Protocol – Cryopreservation of Adherent Cells Growing in Serum-free Medium

Overview

• A serum-free freezing medium is used to preserve cell lines which are maintained in the serum-free growth medium.
• Serum-free freezing medium is nothing but the growth medium supplemented with 5 – 10% DMSO.
• Usually, a 2x freezing medium is prepared which is diluted to 1 x using cell suspension. This reduces the damage to cells caused by DMSO.
• DMSO, a cryoprotectant, protects cells from death by preventing ice crystal formation.

Related posts
Protocol – Thawing and Revival of Cryopreserved Cells

Overview of the procedure

• Harvested cells from monolayer culture/suspension culture are resuspended in the used (conditioned) culture medium at cell density twice the recommended freezing cell density (for example, if the recommended freezing cell density of 5x 10⁵ cells/ml, make cell suspension of 1 x 10⁶ cells/ml).
• A 2x ice-cold freezing medium is added to bring the final concentration 1 x with respect to cell density and freezing medium i.e., 1:1 dilution.
• The cell suspension is aseptically aliquoted into cryogenic vials (1 ml/vial).
• All cryogenic vials are subjected to slow cooling (1 – 3°C/min) overnight in a -80°C freezer.
• The next day, all cryogenic vials are transferred to liquid nitrogen containers or -150°C incubators.

Requirements

Reagents
> 2 x Freezing medium (20% DMSO + complete growth medium)
> PBS (Ca2+-free and Mg2+-free)
> Trypsin EDTA

Equipment and disposables
> Sterile cryogenic vials
> Sterile conical tubes (15-mL or 50-mL)
> Controlled rate freezing apparatus
> Haemocytometer/Trypan Blue
> -80°C freezer
> Liquid nitrogen storage container/-150°C freezer
> Benchtop centrifuge with 45° fixed-angle or swinging-bucket rotor (e.g., Eppendorf™ 5804 Series)
> Personal protective equipment (sterile gloves, laboratory coat, Full-face protective mask/visor)
> Laminar flow hood
> Pipette tips and pipetman
> Serological pipettes and Pipetboy
> Inverted phase-contrast microscope

Starting materials
Late log-phase healthy monolayer culture (90% confluent culture)
Tip: Change the medium the previous day to remove any cell debris present. This will also help you to keep culture healthy and to examine culture for contamination. Moreover, you will use this medium to freeze cells.

Protocol

Prior to start
1. Visually inspect the cell culture carefully under an inverted phase-contrast microscope and make sure that cells are healthy and do not have any contamination. 
2. Properly label Cryogenic vials. You must write clearly the cell line name, passage number, and date on cryogenic vials.
Note: Here we describe a general procedure for cryopreserving adherent cell culture maintained in a serum-free chemically-defined culture medium. For specific details, we recommend you to carefully read the manual provided with the cell line or consult the supplier.

Step 1: Harvest cells from the culture using the standard procedure
> Take out culture medium aseptically and store in a sterile tube (this medium will be used later for resuspension of cell pellet).
> Carefully wash cell monolayer with PBS.
> Use the standard procedure to dislodge cells from culture dishes. Often to dislodge serum-free culture, commercial cell dissociation solutions are used. Add sufficient amounts of cell dissociation solution (1 ml for a T25 flask) and incubate at 37°C for 1 – 2 min. Inspect the dish for cell detachment. Incubate at 37°C for some more time if cells are not dislodged.
> Once the cells are dislodged, tap the flask/dish 2-3 times to make sure all the cells have come out from the dish surface. Immediately add growth medium (4 ml for a T25 flask) and flush the entire surface of the dish by pipetting to collect all cells from the dish. 
> Transfer cell suspension to a sterile centrifuge tube. Cells from two or more dishes from the same passage number (subculture) can be combined in one tube.
> Harvest cells from cell suspension by centrifugation at 4°C for 5 – 10 min at 250 × g (1000 – 1500 rpm for Eppendorf™ 5804 Series benchtop centrifuge).
> Discard the supernatant and resuspend cell pellets in sufficient amounts of conditioned media (e.g., 2 ml).

Note:
Most commercially available cell dissociation agents lose their activity substantially when diluted.

Cautions:
1. Make sure that the dissociation of cells from the surface does not cause enormous cell death. Healthy cells are a prerequisite for cryopreservation.
2. Centrifugation speed should be sufficient to get a soft pellet. Pellet should not be too tight. A tight pellet will be difficult to resuspend and attempts to resuspend it by vigorous pipetting may cause cell death.

Step 2: Determine viable cell density in cell suspension (optional)
> Determine viable cell numbers using trypan blue exclusion assay and a hemocytometer.
> To count viable cells, mix 20 µl cell suspension to 20 µl trypan blue solution. Place the solution onto the hemocytometer chamber.
> Count live and dead cells. Calculate viable cell count per ml and percentage of dead cells.

Tips
1. You can use other sophisticated methods like Countess® Automated Cell Counter or Moxi Flow Kit which can determine viability cell count quickly.
2. In many cases, based on previous experience you can determine how many cryovials can be frozen from a culture dish. One can prepare 2- 4 ml cell suspension in freezing medium from a near confluent T25 flask which can be used to prepare 2 – 4 cryogenic vials (1 ml/vial).
3. Counting is required when cell number is limited and you want to freeze as many cryogenic vials as possible e.g., primary culture.

Cautions
1. Make sure that cells are properly resuspended before counting viable cells.
2. Don’t use cells if cell death is high. In this case, cells can be plated in fresh culture dishes that can be used for cryopreservation when they are ready.

Step 3: Dilute cells 1: 1 in an ice-cold 2x freezing medium to obtain recommended viable cell density  (5 x 10⁵ – 1 x 10⁶ cells/ml)
> Adjust the cell density twice as recommended freezing cell density using the medium collected from the cell culture dish (conditioned medium).
> Now dilute it 1: 1 with 2 x freezing media to obtain the right viable cell density. Add an appropriate volume of ice-cold freezing medium to obtain the right viable cell density (between 1 x 10⁶ – 5 x 10⁶ cells/ml). 
> Resuspend the cells thoroughly with gentle pipetting.

Tips
1. Cell density in the freezing medium can vary with cell lines.
2. Add 2x ice-cold freezing media slowly and mix simultaneously.
3. If the cell suspension is diluted and 2x cell density can not be achieved, centrifuge and resuspend the pellet again in an appropriate volume of conditioned medium.

Cautions
1. Since DMSO can be toxic to cells, it is advisable to use a chilled freezing medium. Try your best to maintain the temperature of cell suspension at 4°C.
2. Quickly resuspend the pellet in the freezing medium immediately after aspirating supernatant.

Step 4: Aliquot cell suspension into cryogenic vials
> Place cryovials on ice.
> Transfer 1 ml aliquots of cell suspension into cryovials.
> Tighten caps on vials immediately.

Caution
While aliquoting, frequently, and gently mix the cells to maintain a homogeneous cell suspension.

Step 5: Subject cryovials to slow cooling (1 – 3°C/min) overnight in -80°C freezer
> Place all cryogenic vials in a controlled rate freezing apparatus (e.g., CoolCell® Cell Freezing Containers from Biocision) and immediately store in -80°C freezer overnight.

Tips
1. The most efficient way to freeze cryogenic vials is to use controlled-rate freezers (e.g., CryoMed™ Controlled-Rate Freezers from ThermoFisher Scientific). However, for most serum grown cell lines, one can use commercially cooling devices e.g., CoolCell® Cell Freezing Containers from Biocision, or  Mr. Frosty™ Freezing Container from ThermoFisher Scientific.
2. Homemade cooling devices – Thermocol box (small size) filled with cotton or tissue paper, can also be used in place of commercial cooling devices.

Step 6: Store cryogenic vials in liquid nitrogen
> Transfer all frozen cryogenic vials to a liquid nitrogen container the next day. Alternatively, frozen cryogenic vials can also be stored in a -150°C freezer.
> Cryogenic vials can be stored in the gas phase or liquid phase of liquid nitrogen.

Tip
A frozen vial can be revived after a few days to ensure that frozen stocks are viable and free of any contamination.

Cautions
1. Biosafety level 2 cell lines should be stored in the gas phase of liquid nitrogen.
2. You must maintain the proper records of the location of frozen cell lines.
3. Wear protective equipment when handling liquid nitrogen. Remember that cryogenic vials may explode if they are stored in a liquid phase of liquid nitrogen.