Cell Line Cryopreservation Protocol

Cryopreservation of cell lines can prevent cell loss due to contamination, minimize genetic changes in passaged cell lines, and avoid aging and transforming limited cell lines. Cell lines should be characterized and checked for contamination before cryopreservation. Cryopreservation media typically contain basal culture medium, cryoprotectants, and protein sources. Cryoprotectants and proteins help cells resist the stress of the freeze-thaw process.

Tab.1 The key materials in cryopreservation.

Materials Needed	Description
Freezing Medium	Commonly 90% FBS, 10% DMSO, or glycerol
70% (volume/volume) alcohol in sterile water	For cell bottle surface disinfection
PBS	without Ca²⁺/Mg²⁺
0.05% trypsin/EDTA in HBSS	without Ca²⁺/Mg²⁺

Preparation
Medium Preparation: Prepare the freezing medium by mixing the complete culture medium with 10% DMSO.
Labeling: Label cryovials with cell line name, passage number, date, and other relevant information.
Harvesting Cells
Inspection: Examine cells under a microscope to ensure they are healthy and at the appropriate confluency (typically 70-80%).
Trypsinization: Detach adherent and semi-adherent cells by treating them with trypsin/EDTA, and resuspend them in a fresh medium in an amount equal to or greater than the volume of trypsin used. Suspension cell lines can be used without additional steps.
Neutralization: Add complete culture medium to inactivate trypsin.
Collection: Transfer the cell suspension to a 15 mL conical tube.
Cell Counting and Centrifugation
Counting: Take a small aliquot of the cell suspension and count cells using a hemocytometer or automated cell counter. For optimal recovery after freezing, cell viability should ideally exceed 90%.
Centrifuge the remaining cell suspension at 1000 rpm for 5 minutes to collect the cells.
Resuspension: Re-suspend cells at 2-4x10⁶ cells per mL in a freezing medium.
Gently remove the supernatant and suspend the cell pellet in a cold freezing medium, adjusting the volume to achieve a final concentration of 1-2 x 10⁶ cells/mL.
Aliquoting and Freezing
Aliquoting: Transfer 1 mL of the cell suspension into each labeled cryovial. Be certain to label each cryovial accurately with the cell line name, passage number, lot number, cell concentration, and date.
Freezing: Place the cryovials into a cryopreservation container for a controlled cooling rate of approximately -1°C per minute. Transfer the container to a -80°C freezer and leave it overnight.
Long-term Storage
Transfer to Liquid Nitrogen: After 24 hours at -80°C, quickly transfer the cryovials to liquid nitrogen storage for long-term preservation.

Notes

Sterility: Maintain sterile conditions throughout the procedure to prevent contamination.
DMSO Handling: Handle DMSO with care, as it can be toxic. Work quickly to minimize cell exposure to DMSO before freezing.
Freezing Rate: Ensuring a slow, controlled freezing rate is critical for cell viability. Rapid freezing can cause ice crystal formation and damage cells.
Thawing: When ready to use the frozen cells, thaw them quickly in a 37°C water bath and promptly dilute them in a fresh culture medium to minimize DMSO exposure.
Viability Check: After thawing, check cell viability and count cells to determine recovery rate.

SERVICE

Creative Biolabs provides detailed cell cryopreservation protocols worldwide. Following this detailed protocol, cells can be effectively cryopreserved and later revived with high viability, ensuring their availability for future experimental use.

All listed services and products are For Research Use Only. Do Not use in any diagnostic or therapeutic applications.

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