Tissue Culture Series #4: Cell Freezing

Summary

This video provides an in-depth guide to the cell freezing process, an essential technique in tissue culturing. The script covers key topics such as the importance of cell freezing for preserving immortalized cell lines, necessary safety precautions, and the materials required, including freezing media and cryovials. The procedure is outlined step by step, from cell preparation and counting to the freezing and storage process. By following these instructions, researchers can ensure the viability of their cell cultures for future use in experiments and studies.

Takeaways

• 😀 Proper safety measures are essential when handling mammalian cell culture and liquid nitrogen.
• 😀 Cryopreservation is crucial for long-term storage of cell lines due to their limited passage numbers and potential genetic changes.
• 😀 Freezing media such as CryoStar or a 90:10 mixture of DMSO and SAR are used to protect cells during freezing.
• 😀 DMSO is a cryoprotectant that protects cells but is hazardous, so handling precautions must be followed.
• 😀 Freezing media should be chilled before use to minimize the exposure of cells to room temperature DMSO.
• 😀 Label cryovials with essential information, including cell type, passage number, date of freezing, and cell concentration.
• 😀 Use an appropriate freezing container (such as isopropanol) to slowly reduce the temperature of the cells at 1°C per minute.
• 😀 Cells should be in the exponential growth phase for optimal freezing results, avoiding overcrowding or confluence.
• 😀 Both adherent and suspension cells can be frozen, but adherent cells must be detached from the culture flask before freezing.
• 😀 For freezing, cells should have greater than 90% viability, and a typical concentration of 1-2 million cells/mL is recommended.

Q & A

What is the significance of cell freezing in tissue culturing?

Cell freezing is a foundational process in cell-based research. It allows the storage of cells at a specific passage before they undergo genetic perturbations due to their immortalization, ensuring they remain viable for future experiments and research.

Why is it important to freeze cells during early passages?

Freezing cells during early passages is crucial because cell lines have limited passage numbers before genetic changes occur that can affect their stability and behavior. Early freezing preserves cells in their original, less-altered state.

What protective equipment is necessary for cell freezing?

Proper protective equipment, such as gloves, safety glasses, and lab coats, should be worn to protect researchers from risks associated with handling mammalian cell cultures and liquid nitrogen, which can be hazardous.

What is cryoprotectant and why is DMSO used in cell freezing?

A cryoprotectant is a substance that helps prevent cell damage during freezing. DMSO (Dimethyl sulfoxide) is commonly used because it readily diffuses through cell membranes and prevents ice crystal formation inside the cells, which could otherwise damage them during freezing.

What are some commercial freezing media options available for cell preservation?

CryoStor is a commercial animal-component-free reagent often used for freezing cells. It is available in different formulations based on DMSO concentration to suit various cell types.

How should freezing media be prepared and handled?

Freezing media should be chilled before use to minimize exposure to DMSO at room temperature. It’s also important to label the cryovials properly with details like cell type, passage number, date of freezing, and cell concentration.

What role does isopropanol play in the freezing process?

Isopropanol is used in a freezing container to gradually reduce the temperature of the cells at a controlled rate (1°C per minute). This slow cooling helps minimize cell damage during the freezing process.

What are the conditions under which cells should be frozen for optimal preservation?

Cells should be in their exponential growth phase, not overly confluent or crowded, and must have greater than 90% viability at the time of freezing to ensure high-quality, viable frozen stocks.

How should adherent cells be prepared for freezing?

Adherent cells must first be detached from their culture flask using trypsin or other methods. After detaching, the cells are resuspended in culture media, counted, and assessed for viability before freezing.

What is the proper procedure for freezing cells?

The general procedure involves centrifuging the cells, carefully aspirating the media, and resuspending the cell pellet in chilled freezing media. The cells are then aliquoted into cryovials and placed in a freezing container that slowly reduces the temperature before being transferred to liquid nitrogen for long-term storage.