Cell culture techniques 4 - Cryopreservation

Summary

This video from Shamus Biology's animal cell culture series focuses on cryopreservation, a critical process for preserving cells for future research. It explains the importance of cryopreservation in reducing contamination risks and maintaining cell integrity. The script details the steps for preparing cells during the log phase, using cryoprotectants like DMSO to prevent ice crystal formation, and the gradual freezing process to ensure cell viability post-thawing.

Takeaways

• 🧊 Cryopreservation is essential for preserving cell cultures to prevent waste of time and resources after extensive experimentation.
• 🛡️ Cryopreservation reduces the risk of microbial contamination, cross-contamination with other cell lines, and genetic drift or morphological changes due to the cessation of cellular activity at low temperatures.
• 🌡️ Cells should be cryopreserved during the log phase of growth when they are most viable, avoiding the plateau phase where viability decreases.
• 🔬 The process of cryopreservation begins with passaging the cells and ensuring they are in a healthy, viable state before preservation.
• 🌀 After passaging, cells are centrifuged to remove debris and resuspended in a serum-containing media to prepare for cryopreservation.
• 🧪 DMSO (dimethyl sulfoxide) is used as a cryoprotectant to prevent the formation of ice crystals that could damage or kill the cells during freezing.
• 🔬 The exact mechanism of how DMSO protects cells is not fully understood, but it is known to be effective in cryopreservation.
• 💧 The addition of 10% DMSO in Fetal Calf Serum (FCS) is a common method to prepare cells for cryopreservation.
• 🧊 Cells are first frozen at -80°C before being transferred to liquid nitrogen at -196°C to ensure a controlled and slow freezing process.
• ⏱ The gradual decrease in temperature is crucial for maintaining cell integrity and morphology, avoiding rapid changes that could harm the cells.
• 📚 Understanding the different stages of cell culture and growth phases is important for effective cryopreservation and should be reviewed for optimal results.

Q & A

Why is cryopreservation of cells important in cell culture processes?

Cryopreservation is important because it allows the storage of cells for future use, reducing the risk of microbial contamination, cross-contamination with other cell lines, and genetic drift or morphological changes. It ensures that valuable cell properties are preserved for future research.

How does cryopreservation reduce the risk of microbial contamination?

Cryopreservation reduces the risk of microbial contamination because cells are stored at very low temperatures, where microbial activity is significantly reduced or stopped.

What are the benefits of storing cells at low temperatures?

Storing cells at low temperatures reduces the risk of microbial contamination, cross-contamination with other cell lines, genetic drift, and morphological changes. It also preserves the cells' viability for future use.

What is the significance of the log phase in the cell culture growth cycle for cryopreservation?

The log phase is significant because cells are most viable during this phase. Storing cells from the log phase ensures higher viability when they are thawed and used in future research.

What steps are involved in the cryopreservation process after cell passaging?

After cell passaging, the steps involved are: checking for the log phase, resuspending the cells in serum-containing media, centrifugation to remove debris, resuspending cells in 10% DMSO in FCS, transferring the cells to cryovials, freezing at -80°C, and then transferring to liquid nitrogen at -196°C.

Why is DMSO used in the cryopreservation process?

DMSO is used because it acts as a cryoprotectant, helping to prevent the formation of ice crystals inside the cells, which can cause cell death by cracking the cell open.

What is the purpose of freezing cells at -80°C before transferring to liquid nitrogen?

Freezing cells at -80°C before transferring to liquid nitrogen is done to slowly reduce the temperature, preventing rapid changes that could damage the cells. This gradual reduction helps maintain cell integrity.

What is the final temperature used for long-term cell storage in cryopreservation?

The final temperature used for long-term cell storage in cryopreservation is -196°C, typically achieved using liquid nitrogen.

What potential problems does cryopreservation help to avoid?

Cryopreservation helps to avoid microbial contamination, cross-contamination with other cell lines, genetic drift, morphological changes, and the formation of damaging ice crystals within cells.

What are the key properties of cells that cryopreservation helps to maintain?

Cryopreservation helps to maintain the cells' viability, genetic stability, and morphological integrity, ensuring that the cells remain suitable for future research.