Protocol: Cryopreservation of Suspension Cell Culture

OVERVIEW

Freezing suspension cell cultures is generally easier than freezing adherent cultures, as it eliminates the need for trypsinization. However, in cases where large cell clumps are present, trypsinization may be necessary to improve yield upon revival.

To cryopreserve a suspension culture, cells are first harvested by centrifugation and the resulting pellet is gently resuspended in ice-cold freezing medium at the appropriate cell density. The resuspension is then aliquoted into cryovials and subjected to slow freezing overnight before transferring to the liquid nitrogen or a -150°C freezer.

REQUIREMENTS
Reagents
♦ Growth medium (e.g., DMEM supplemented with 10% FBS)
♦ Freezing medium (70% growth medium + 20% FBS + 10% DMSO)

Equipment and disposables
♦ Sterile cryogenic vials
♦ Sterile conical tubes (15 mL or 50 mL) (e.g., SARSTEDT, 62.554.502 or 62.547.254)
♦ 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

Prior to start:
1. Inspect the cell culture under the inverted phase-contrast microscope to ensure cells are healthy and free of contamination.
2. Label cryogenic vials clearly with cell line name, passage number, and date.

Note: This protocol provides a general procedure; for specific details, refer to the manual from the cell line supplier.

Tip: For optimal cryopreservation, It is recommended to change the medium of the cells the day before freezing. This involves harvesting cells via centrifugation, resuspending the pellet in fresh growth medium, and culturing under appropriate conditions.

Precaution: Always wear appropriate personal protective equipment (lab coat, gloves, goggles, etc) and follow institutional guidelines.

PROTOCOL

Step 1: Determine viable cell density in cell suspension (optional)
◊ Suspend the suspension culture properly by pipetting a few times up and down. This will also break the clumps.
◊ Determine viable cell numbers using trypan blue exclusion assay and a hemocytometer. Briefly, to count viable cells, mix 20 µl cell suspension with 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.
◊ Viability over 90% is considered a healthy culture.

Note: This step is optional. Researchers with experience and familiarity with the specific cell line can assess cell health and visually approximate cell density using a microscope, allowing them to proceed directly to Step 2. For cryopreserving most mammalian tumor cell lines, precise cell counts per cryogenic vial are not essential unless there is a scarcity of cells and you want to freeze as many vials as possible with the minimum sufficient number of cells per vial. A range of 1 x 10⁶ to 5 x 10⁶ cells/vial is generally acceptable.

Tips
1. Automated cell counters like Countess® or Moxi Flow can provide quicker assessments.
2. In many cases, based on previous experience you can determine how many cryogenic vials can be frozen from a culture dish.

Precautions:
1. Ensure proper resuspension before counting.
2. If cell death is high, do not cryopreserve. Instead, harvest cells by centrifugation, resuspend in growth medium, culture for one day, and reassess viability before considering cryopreservation.

Step 2: Harvest cells from the culture
◊ 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.
◊ Centrifuge at 4°C for 5–10 min at 250 × g (1000 – 1500 rpm for Eppendorf™ 5804 Series benchtop centrifuge).
◊ Carefully aspirate supernatant as much as possible without disturbing the cell pellet.
◊ Flick the tube with your finger several times to dislodge the pellet.

Precaution: The centrifugation speed should be sufficient to form a soft pellet without losing cells. The pellet should not be too tight, as a tight pellet will be difficult to resuspend and can lead to cell clumps. Attempts to resuspend and break up clumps by vigorous pipetting may cause cell death. (Read more: Poor Trypsinization and Cell Clumps: Impact on Cryopreservation)

Step 3: Resuspend the cells in freezing medium at the recommended 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.

Tip: Cell density in the freezing medium can vary with cell lines. In most cases, high cell density is good for cell recovery.

Precautions
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 the supernatant.

Step 4: Aliquot cell suspension into cryogenic vials
◊ Place the cryogenic vials on ice to keep them chilled and maintain cell viability during the transfer process.
◊ Transfer 1 ml aliquots of the cell suspension into each cryogenic vial.
◊ Immediately tighten the caps on the vials to prevent contamination.

Tip: 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 a -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 commercial cooling devices e.g., CoolCell® Cell Freezing Containers from Biocision, or  Mr. Frosty™ Freezing Container from ThermoFisher Scientific.
2. Homemade cooling devices – A insulated box such as 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 2 weeks to ensure that frozen stocks are viable and are free of any contamination.

Precautions
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.

Disclaimer: While we strive to provide accurate and up-to-date information, we cannot guarantee its absolute accuracy or completeness. Please consult additional sources for verification.