Safeguarding your samples throughout the entire cryopreservation process can be tricky and even unproductive, thanks to protocols that unknowingly undermine your results. Understanding the seven keys to protect your cryopreserved cells – from selecting the right storage temperatures and proper cooling methods to avoiding common safety hazards when using liquid nitrogen – can equip any lab with the knowledge necessary to ensure sample safety and optimal cryopreservation results.
Thermo Fisher Scientific's on-demand webinar, The Seven Keys to Safeguarding Your Cryopreserved Cells, explores the many variables involved in planning for cryopreservation in your cell culture process. Let's explore these steps and unlock the critical information to ensure high cell viability.
1. The Cryopreservation Workflow
The cryopreservation workflow shows the process from cell culture to cryostorage at recommended temperatures. All critical elements which can impact cell viability include:
- Selection of the optimal temperature based on cell type
- Choosing the correct cryogenic tubes
- Controlling the cooling rate correctly
- Expanding the storage system to expand storage capacity
- Tracking and organizing samples with bar code management software
- Using suggested protection for samples in liquid nitrogen
If cells are properly preserved there will be high cell viability through thawing and re-culturing. When using the recommended protocol and products, consistent results can be achieved in terms of cell viability. In general, a successful cryopreservation process should result in healthy cells with a viability over 90% upon thawing and there should be no sign of microbial contamination. However, when using the wrong protocol and products disasters may happen, leading to the loss of valuable cells.
2. Ensuring the Right Temperatures
Storage of cells for use in downstream cell culture requires that cellular biological activity is suspended in such a manner that enzymatic or chemical activity, which may cause damage to the material in question, is halted. When the temperature is below -132⁰C biological activity is effectively stopped. However, the most desirable temperature range for the long-term storage of cells is -150⁰C through -196⁰C. There are three common methods for cryopreservation:
- Electric (-135⁰C) freezer: There are several advantages to this freezing method including ease of maintenance, steady temperature of -135⁰C, and low running costs. However, it also requires liquid nitrogen back-up in case of power or mechanical failure.
- Liquid phase of liquid nitrogen: This method can achieve the lowest possible temperature which is -196⁰C. While this method is mechanically reliable, when compared to the electric freezer method, it costs significantly more. Most importantly, it requires proper protection when submerging the samples directly into the liquid nitrogen to ensure there isn't any cross-contamination.
- Vapor phase of liquid nitrogen: This is the recommended method for long-term storage of cells. It achieves the desirable low temperatures without the risk of cross-contamination. However, the temperature has the potential to fluctuate between -135⁰C and -190⁰C.
3. The Cooling Rate is Critical!
In the early 1950s studies found that rapid immersion of samples such as embryos, bone marrow, and stem cells in liquid nitrogen did not produce the necessary viability to make them usable after thawing, suggesting that rapid cooling could cause serious damage to cultured cells. It was also discovered that when the temperature drops below 0⁰C during the freezing process, ice crystals begin to form in an intracellular environment which increases the solute concentration of the cultured media. As a result, water begins to move out of the cells and into the partially frozen media, beginning the process of cellular dehydration and shrinkage. Cell permeability affects the rate of water loss; more permeable cells are able to tolerate rapid cooling better than less permeable cells. With few exceptions, a cooling rate that is close to 1⁰C per minute is preferred. This helps minimize cell damage caused by either a fast cooling rate or a slow cooling rate.
The final four keys include:
4. Selecting the correct cryovials
5. Understanding the risks
6. Dense Storage: doubling the capacity of your freezers
7. Sample Organization and Tracking
To unlock the answers to the final 4 keys to safeguard your cryopresereved cells, download the webinar below!