Protocol – Cryopreservation of Adherent Cell Culture

Overview

Cryopreservation of adherent cell cultures requires detaching the cells from the substratum, typically through trypsinization. The detached cells are collected by centrifugation, then gently resuspended in an appropriate freezing medium at the right volume to achieve the desired cell density. The cell suspension is aliquoted into cryogenic vials and subjected to a controlled slow-freezing process overnight to ensure optimal cell viability. Finally, the vials are transferred to long-term storage in liquid nitrogen or a -150°C freezer.

Note: Here, we provide a general procedure for cryopreserving adherent cell cultures. For cell line-specific protocols, we strongly recommend reviewing the manufacturer’s manual or consulting the supplier to ensure optimal preservation and viability.

Protocol-Cryopreservation of Adherent Cell CultureRequirements

Reagents
♦ Growth medium (e.g., DMEM supplemented with 10% FBS)
♦ PBS (Ca2+-free and Mg2+-free)
♦ Trypsin-EDTA solution (or a suitable cell dissociation reagent)
♦ Freezing medium (50% 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

Starting materials
Late log-phase healthy monolayer culture (80% – 90% confluent culture)

Tip: Change the medium the previous day to remove any cell debris present. This will also help you to keep the culture healthy and to examine the culture for contamination.

Protocol

Prior to start
1. Carefully inspect the cell culture under an inverted phase-contrast microscope to ensure that the cells are healthy and free of contamination.
2. Properly label the cryogenic vials. Clearly write the cell line name, passage number, and date on each vial.

Step 1: Harvest cells from the culture using a standard procedure (trypsinization)

  • Discard the culture medium and wash the monolayer with PBS. Add an adequate amount of trypsin-EDTA solution (1–2 ml for a T25 flask) and incubate at 37°C for 1–2 minutes. Inspect the dish under the microscope for cell detachment. If the cells are not dislodged, continue incubating at 37°C for additional time.
  • Once the cells are dislodged, tap the flask or dish 2-3 times to ensure all cells have come off the surface. Add serum-containing growth medium (4 ml for a T25 flask) and gently flush the entire surface by pipetting to collect all cells from the flask.
  • Transfer the cell suspension to a sterile centrifuge tube. Cells from two or more dishes with the same passage number can be combined in one tube.

Note:
Serum in the complete growth medium has trypsin inactivating activity.

Tip:
At this stage, cells can be collected by centrifugation, and the resulting cell pellet can be resuspended in an adequate amount of growth medium. However, this is optional. If you suspect significant cell death during trypsinization or if the cell suspension is too diluted, centrifugation and resuspension can be performed.

Caution:
Ensure that the trypsinization process does not lead to excessive cell death, as healthy cells are essential for successful cryopreservation.

Step 2: Determine viable cell density in cell suspension (optional)

  • Determine viable cell numbers using trypan blue exclusion assay and a hemocytometer. This method involves mixing a small aliquot of the cell suspension with trypan blue dye, which stains dead cells. By counting the unstained (viable) cells under the microscope, you can accurately assess cell viability and density.
  • To count viable cells, mix 20 µl of the cell suspension with 20 µl of trypan blue solution, then place the mixture onto the hemocytometer chamber.
  • Count the live and dead cells. Then, calculate the viable cell count per mL and the percentage of dead cells.
  • Viability over 90% is considered a healthy culture.

Tips
1. You can use other advanced sophisticated methods, such as the Countess® Automated Cell Counter or the Moxi Flow Kit, which can quickly determine viable cell counts.
2. If the cells are very diluted, collect them by centrifugation and resuspend the cell pellet in an appropriate volume of growth medium.
3. In many cases, based on previous experience, you can estimate how many cryovials can be frozen from a culture dish. From a near-confluent T25 flask, you can prepare a 2-4 mL cell suspension in a freezing medium, which can then be used to fill 2 to 4 cryogenic vials (1 mL per vial).
4. Counting is essential when the cell number is limited, such as in primary cultures, and you want to maximize the number of cryogenic vials for freezing.

Cautions
1. Ensure that the cells are properly resuspended before counting the viable cells.

2. Avoid using cells if there is a high rate of cell death. In this case, the cells can be plated in fresh culture dishes and can be used for cryopreservation when they are ready.

Step 3: Resuspend the cells in ice-cold freezing medium at the recommended viable cell density of (1 x 106 – 5 x 106 cells/ml)

  • Harvest the cells from the cell suspension by centrifugation at 4°C for 5 to 10 minutes at 250 × g (1000 – 1500 rpm for the Eppendorf™ 5804 Series benchtop centrifuge).
  • Carefully aspirate the supernatant as much as possible without disturbing the cell pellet.
  • Flick the tube with your finger several times to dislodge the pellet.
  • Add an appropriate volume of ice-cold freezing medium to achieve the desired viable cell density (between 1 x 106 – 5 x 106 cells/ml). Resuspend the cells thoroughly using gentle pipetting.

Tip
Cell density in the freezing medium can vary by cell line. In most cases, a higher cell density is beneficial for cell recovery.

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 the supernatant.
3. Centrifugation speed should be sufficient to achieve a soft pellet. The pellet should not be too compact, as a tight pellet can be difficult to resuspend. Vigorous pipetting attempts to resuspend a tight pellet may result in cell death.

Step 4: Aliquot the cell suspension into cryogenic vials.

  • Place the cryovials on ice.
  • Transfer 1 mL aliquots of the cell suspension into the cryovials.
  • Tighten the caps on the 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 store immediately in a -80°C freezer overnight.

Tips
1. The most efficient way to freeze cryogenic vials is by using controlled-rate freezers (e.g., CryoMed™ Controlled-Rate Freezers from ThermoFisher Scientific). However, for most serum-grown cell lines, you can use commercial cooling devices, such as CoolCell® Cell Freezing Containers from Biocision or Mr. Frosty™ Freezing Containers from ThermoFisher Scientific.
2. Homemade cooling devices: A small Thermocol box filled with cotton or tissue paper can be used as an alternative to 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, they can also be stored in a -150°C freezer.
  • Cryogenic vials can be stored in either the gas phase or the liquid phase of liquid nitrogen.

Tip
A frozen vial can be revived after two weeks to confirm that the frozen stocks are viable and free from contamination.

Caution
1. Biosafety Level 2 cell lines should be stored in the gas phase of liquid nitrogen.
2. It is important to maintain accurate records of the locations of frozen cell lines.
3. Wear protective equipment when handling liquid nitrogen. Be aware that cryogenic vials may explode if stored in the liquid phase of liquid nitrogen.

 

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