Cryopreservation refers to the cooling of cells to a point at which activities inside the cells cease. When done effectively, cryopreservation freezes cells in a way that minimizes damage, so that cell viability is high after storage and subsequent thawing. Freezing cells can also limit contamination and reduce genetic changes. A cryoprotectant is added during cell freezing to minimize damage from ice crystals and osmotic changes that would occur in their absence.
Materials for cell freezing
The following materials are used for freezing cells:
- Cells prepared to be frozen
- Cell freezing media, often a cell culture medium with additives
- Cryoprotective agent, such as dimethyl sulfoxide (DMSO)
- Cryovials or other containers suitable for long-term storage
- A method for freezing and a freezing chamber
- A liquid nitrogen (N2) tank for long-term storage
- Serum products like fetal bovine serum (FBS) – optional
How to freeze cells
The methods vary depending on the cell type and eventual use after thawing. For research applications with mammalian cells including human cell types, here’s a simple how-to list:
1. Passage cells
Use healthy and actively growing cells – in a logarithmic growth phase – that haven’t been passaged many times. Best practice is to passage cells 24 to 48 h before freezing.
2. Prepare cells
After passaging your cells, remove them from the growth vessel. For adherent cells, this may involve the use of a cell dissociation reagent; in this case be sure to neutralize the reagent to prevent excess cell damage. Pool all the cells and subject them to centrifugation. Remove and discard the supernatant under aseptic conditions.
3. Suspend cells
Freezing cells may have detrimental effects on the cells. To mitigate damage like osmotic stress, membrane damage, or ice crystal formation, be sure to use cell freezing media with a cryoprotectant like DMSO. Some methods use serum, but serum-free is preferred for cell therapy applications. Dispense the cell suspension into cryovials, cryobags, or other appropriately sized containers suitable for your cell volume.
4. Freeze cells
Freeze the cells using liquid nitrogen, a passive freezer, or a controlled-rate freezer. Once you complete the freezing process, move your frozen cryovials or containers to a liquid nitrogen tank for storage.
How to thaw cells
To thaw cells, remove cryovials from the tank and place them in a water bath or dry automated thawing system. Once thawed, transfer cells to prewarmed cell culture medium. Inspect cells after 24 h to determine health status and viability.
Cell freezing media for cell culture FAQs
Below are answers to some frequently asked questions about cell freezing media.
What is cryopreservation and cell freezing medium?
Cryopreservation media, also called cell freezing media, are formulations that contain a base medium and a cryopreservative – which may include proteins – to protect cells from the stress caused by the freezing and thawing process.
Why do we cryopreserve cells?
Cryopreservation helps store valuable cells for research or biomanufacturing to preserve viability, minimize contamination, and reduce genetic changes. The goal with cryopreservation is to maintain the cells in a state that gives high viability (% live cells) after thawing and growth in cell culture media.
How do you freeze stem cells?
This process is similar to freezing other mammalian cell types. However, it’s important to use specific cryopreservation media to ensure high viability and to maintain the cells’ potential to differentiate.
Cytiva’s stem cell cryopreservation medium is designed for use with neural progenitor or stem cells, embryonic stem cells (ESCs), and induced pluripotent stem (iPS) cells.