Freezing cryovials is a crucial step in preserving biological samples for future use. These small plastic containers are specially designed to store cells, tissues, or other biological materials at ultra-low temperatures. To ensure that your samples remain viable during freezing and storage, it’s important to follow proper procedures. Let’s explore the key steps to safely freeze cryovials.
Step 1: Prepare Your Samples
Before freezing your samples in cryovials, make sure they are prepared and ready for long-term storage. Depending on the type of sample (cells, tissue, etc.), there are different preparation techniques:
- Cell samples: Cells need to be suspended in a cryoprotectant solution. Cryoprotectants like dimethyl sulfoxide (DMSO) or glycerol help protect the cells from damage during freezing.
- Tissue samples: Larger tissue samples may need to be cut into smaller pieces to allow for even freezing.
- Protein or DNA/RNA samples: Make sure these are in appropriate buffers to prevent degradation during freezing.
Always use sterile techniques to avoid contamination.
Step 2: Choose the Right Cryovials
It’s essential to use high-quality cryovials that can withstand freezing temperatures without breaking or leaking. Look for cryovials made from materials like polypropylene, which is durable and able to handle extreme cold.
Cryovials come in different sizes, ranging from 1 mL to 5 mL, so choose the size that fits your sample volume. Additionally, opt for cryovials with secure, leak-proof caps and clearly marked labels to help you identify the samples later.
Step 3: Label the Cryovials
Proper labeling is vital when freezing cryovials. Use labels that can withstand ultra-low temperatures and clearly write the sample name, date, and any other identifying information. Many cryovials come with pre-labeled caps or stickers, but you can also use a permanent marker or specialized labeling system.
Avoid using ordinary ink pens or labels, as they may smudge or fall off under freezing conditions.
Step 4: Add Cryoprotectant
Adding cryoprotectant is a critical step to prevent damage to your samples during freezing. Cryoprotectants, such as DMSO or glycerol, help protect the cells by preventing the formation of ice crystals, which can damage cell membranes.
For cell samples, mix the cryoprotectant into the cell suspension just before freezing. Be sure to follow the correct concentration guidelines, typically around 5-10% of the total volume. Once the cryoprotectant is added, gently mix the solution to ensure even distribution.
Step 5: Aliquot the Samples into Cryovials
Once the samples are prepared and mixed with cryoprotectant, carefully transfer them into the cryovials. Use a pipette or sterile transfer tool to aliquot the samples into the cryovials without spilling or contaminating the samples.
Make sure not to overfill the cryovials. Leave a small amount of air space at the top to allow for expansion when the sample freezes. This helps prevent the vial from cracking under pressure.
Step 6: Gradual Freezing Process
The freezing process must be done gradually to prevent cell damage from ice crystal formation. The recommended method is to freeze samples slowly, usually at a rate of -1°C to -3°C per minute, down to a temperature of about -80°C.
Here’s how to achieve this:
Using a Controlled-Rate Freezer: This is the best option if available. It allows precise control over the freezing rate, ensuring gradual and consistent freezing.
Using an Isopropanol Freezing Container: If you don’t have access to a controlled-rate freezer, you can use a specialized freezing container filled with isopropanol. These containers can mimic the gradual freezing process when placed in a -80°C freezer.
After reaching -80°C, the samples are ready for long-term storage.
Step 7: Transfer to Liquid Nitrogen Storage
Once the cryovials are frozen at -80°C, they can be transferred to liquid nitrogen storage for long-term preservation. Liquid nitrogen storage provides ultra-low temperatures of around -196°C, which is ideal for preserving the viability of biological samples over long periods.
Here’s how to do it safely:
- Wear protective gear: Always wear gloves, face shields, and appropriate clothing when handling liquid nitrogen.
- Minimize exposure: Limit the amount of time the cryovials are exposed to air before placing them into liquid nitrogen to prevent temperature fluctuations.
- Store cryovials in cryoboxes: Cryovials should be placed in organized cryoboxes to ensure easy identification and retrieval later.
Step 8: Documentation and Monitoring
Proper documentation is essential when freezing cryovials. Record details such as the sample type, date of freezing, and storage location. This helps track the samples and ensure proper storage conditions.
Additionally, regular monitoring of your storage system is important. Check that the freezer or liquid nitrogen tank is maintaining the appropriate temperature to prevent sample degradation.
FAQ
1. What are cryovials used for?
Cryovials are used to store biological samples, such as cells, tissues, or DNA, at ultra-low temperatures for long-term preservation.
2. What type of material are cryovials made of?
Cryovials are typically made from polypropylene, a durable plastic that can withstand freezing temperatures without breaking or cracking.
3. Why is cryoprotectant necessary when freezing samples?
Cryoprotectants, like DMSO or glycerol, help protect the cells from damage caused by ice crystal formation during the freezing process. They prevent cell membranes from breaking.
4. How should I label cryovials before freezing?
Use labels that can handle ultra-low temperatures and include details such as sample name, date, and other identifying information. Permanent markers or specialized labels are recommended.
5. Can I freeze cryovials at -20°C?
While -20°C freezers can slow down biological activity, they are not suitable for long-term preservation. Samples should be frozen at -80°C or lower, and ideally stored in liquid nitrogen at -196°C.
6. What is the gradual freezing process, and why is it important?
Gradual freezing involves lowering the temperature slowly, usually at a rate of -1°C to -3°C per minute. This prevents ice crystals from damaging the cells.
7. What tools can I use for gradual freezing?
You can use a controlled-rate freezer or an isopropanol freezing container to freeze samples gradually. Both methods help avoid cell damage during the freezing process.
8. How do I transfer cryovials to liquid nitrogen storage?
Wear protective gear, minimize exposure to air, and use cryoboxes to organize and safely store the vials in liquid nitrogen, which maintains a temperature of -196°C.
9. How long can cryovials be stored in liquid nitrogen?
When stored properly in liquid nitrogen, cryovials can preserve samples for many years, even decades, without significant degradation.
10. How should I document the freezing process?
Keep detailed records, including sample type, freezing date, and storage location. Regularly monitor the storage conditions to ensure proper preservation.