Optimal Freeze Protocol for Cryopreservation of Cultured Cells

Freezing of Cells

Freezing down cells is a great way to maximize research using precious cells for safe long-term storage. However, without proper cryopreservation techniques, the freezing process can be quite traumatic to cells and can sacrifice cell viability and functionality after thawing. To eliminate these risks, it is important to maintain a consistent freeze protocol for cells. While the specific freeze protocol can vary based on the cell type, below is a general guideline of what you need to know for best practice. Your cell culture supplier typically provides specific details.

Freeze Protocol for Cells

Before Starting

Remember to always wear personal protective equipment. Always use aseptic techniques, sterilize all equipment and lab materials that come into contact with the cells, and work inside a laminar flow hood.

Media Exchange

Before freezing down, cells need to be spun down, washed, and re-suspended in a cryogenic vial with a proper freeze media containing a cryoprotective agent, such as DMSO or glycerol. The freeze media can vary depending on the cell type.

Freeze Process

The cooling rate during freezing can affect the outcome. Therefore, cells should be frozen down to -80°C in a controlled rate freezing apparatus. If the drop in temperature occurs too quickly, there is an increased risk of intracellular ice formation, which can physically rupture the cell membrane. However, if the rate of cooling is too slow, there is a risk of chemical toxicity because of higher concentrations of salts.

The optimal freeze velocity is typically around -1°C/min, however, the optimal rate can also vary between cell types depending on several factors, like membrane permeability and surface-to-volume ratio. Ultimately, the optimal rate can be determined empirically and performed consistently with a controlled rate freezer. Some controlled rate freezers require liquid nitrogen, which can be difficult to work with, as well as risk for mycoplasma contamination. Liquid nitrogen-free options like the Strex CytoSensei Controlled Rate Freezer can be a convenient tool for freezing down cells.

Alternatively, the cryovials containing the cells can be placed in an isopropanol chamber or insulating foam and left overnight in a -80°C freezer. The alcohol chamber or insulating foam attempts to estimate a -1°C/min freeze rate by slowing heat transfer, but this method often leads to inconsistent results.

Storage

Once frozen down, cryogenic vials containing the cells are quickly transferred to a  -80°C freezer or liquid nitrogen. Vials can be transferred on dry ice as cell suspensions are quickly thawed at room temperature.