Experimental Protocol for Cryopreservation of Mammalian Cell Lines

Cells are an important tool in biological experiments, and cryopreservation is an important way to preserve cells and maintain their viability for months or years. Cryopreservation minimizes genetic variation while avoiding losses due to contamination. We have an extensive cell bank and can also develop stable cell lines for you according to your needs. In addition, we offer a comprehensive cell culture service that can guarantee the accuracy and scientific validity of your research.

Below are the general procedures for cell cryopreservation.

Preparation

1. Materials required

1–2mL cryovials
Freezing unit
Freezing media
hemocytometer

2. Determination of freezing medium

Before starting, check the information of the frozen cells to determine the correct freezing medium.

Procedures

Label the date, researcher's name, cell number, passage number, and cell type (and any other useful information, such as genetic modifications) on the cryotube.
If the cells are apposed, remove the cell medium, then wash the cells with PBS, add enough trypsin to cover them, and incubate for approximately 37 minutes in a 2°C incubator. Resuspend in cell culture medium and transfer to a 50 mL Falcon tube.
If the cells are in suspension, simply transfer the desired volume directly into a 50 mL Falcon tube.
Count cells with a hemocytometer to determine cell viability. Do not freeze until cell viability is higher than 75%.
Centrifuge for 5 min at 1,000 rpm at room temperature.
Prepare freezing media. The correct freezing medium is determined by the cells being frozen; DMSO is not suitable for all cell types, therefore, glycerol is an alternative. The different types of freezing media used for mammalian cell lines are shown in Table 1.
Remove the supernatant (keep it well since the frozen medium requires it, see Table 1) and gently loosen the precipitate.

Table 1. The different types of freezing media used for mammalian cell lines.

Culture Type	Freezing media	Notes
Cells cultured in FBS-containing media	90% FBS, 10% DMSO	Mix and warm to 37°C before use.
Cells cultured in serum-free media	90% conditioned media, 10% DMSO	Use the supernatant from the centrifuge (see step 7). Mix and warm to 37°C before use.
Cells that require glycerol for freezing	90% FBS, 10% glycerol	Mix and warm to 37°C before use.

Add freezing medium to the desired cell density. For mammalian cells, this is typically 1,000,000/mL of freezing medium. Cells in the freezing medium should not be left at room temperature for more than 10 minutes.
Aliquot 1 mL into cryovials and secure the lids.
Transfer the cryovials to a CoolCell (at room temperature) and then placed them in a freezer at -80°C. The CoolCell will ensure that the temperature drops at a steady rate of 1°C per minute.
After approximately 24 hours, remove the cryovials from the CoolCell and transfer them to liquid nitrogen for long-term storage.

Thawing frozen cell lines

Remove the cryovials from the liquid nitrogen and place them in a 37°C water bath until about 80% thawed (do not thaw at room temperature). The process should take no more than 1 minute.
Use a pipette to transfer the contents of the vial to a 15 mL Falcon tube that contains approximately 10 mL pre-warmed culture medium.
Centrifuge at 500-1000 rpm for 5 min, discard the supernatant, and resuspend in an appropriate amount of cell culture medium.
Transfer the cells to a culture vessel and place it into a 37°C incubator.

Disclaimer

This procedure should be used only as a guide. Please note that Creative Biolabs cannot guarantee the activity of the client's cryopreserved mammalian cells.

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

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