Tissue Culture Series #1 - How to Thaw Cells with High Efficiency

Summary

This video focuses on the critical process of thawing cells from a cryopreserved state for successful cell culturing. Jim Park emphasizes the importance of proper safety equipment, handling techniques, and the careful thawing process, which involves gradual temperature control and media formulation. The video outlines the steps to avoid damaging cells, including slow media addition to prevent osmotic shock and the removal of DMSO to maintain cell integrity. It also covers the necessity of monitoring cell growth and possible causes for low yields, such as poor-quality frozen stocks or improper thawing and storage conditions.

Takeaways

• 😀 Always wear proper protective equipment when handling mammalian cell cultures and liquid nitrogen.
• 😀 Cryopreserved cells are stored with cryoprotectants like DMSO, which require careful handling to avoid skin exposure.
• 😀 Thawing cells should be done quickly, ensuring they are not fully submerged in the water bath to avoid contamination risks.
• 😀 The thawing process should take approximately 90 seconds to prevent ice crystals from damaging the cells.
• 😀 Media used for thawing should be pre-warmed to 37°C, freshly made, and sterilized for optimal results.
• 😀 Add thawing media dropwise to prevent osmotic shock, allowing DMSO to diffuse out of the cells gradually.
• 😀 Avoid vortexing during mixing to prevent damaging delicate cells; inversion is the preferred method.
• 😀 Centrifugation should be done with low speed and longer spin times for delicate cells to minimize damage.
• 😀 The washing step removes DMSO, which could otherwise compromise the cell membrane integrity.
• 😀 Seeding density should be carefully controlled, as sparse seeding slows growth, and overly dense seeding can cause overcrowding and cell death.
• 😀 Regular monitoring of cells is essential after seeding, with media changes often required due to high cell deaths during initial stages.

Q & A

Why is proper protective equipment necessary when thawing cells?

Proper protective equipment is essential to safeguard against risks associated with handling mammalian cells and liquid nitrogen. This includes potential exposure to hazardous materials like DMSO, a cryoprotectant used during freezing.

What is DMSO, and why is it used in the freezing media?

DMSO (Dimethyl Sulfoxide) is a cryoprotectant compound that prevents ice crystal formation within cells during freezing, which can damage the cells. It readily diffuses through the skin, so handling requires caution.

How should the cell vial be thawed after removal from liquid nitrogen?

The vial should be transferred quickly to a 37°C water bath, but not submerged completely. Thawing should take approximately 90 seconds, and the vial should be kept still to avoid agitation, which can cause ice crystals to damage the cells.

What is the purpose of slowly adding culture media to the thawed cells?

Slowly adding culture media to the thawed cells helps prevent osmotic shock. The freezing media is hypertonic, and the gradual addition of fresh media allows the DMSO to diffuse out without causing cell damage.

Why should cells not be vortexed during the thawing process?

Vortexing can damage cells, particularly delicate ones, by causing mechanical stress and disrupting their membranes. Cells should be mixed by gentle inversion to maintain their integrity.

What role does the centrifugation step play after thawing cells?

Centrifugation helps pellet the cells and separate the freezing media (containing DMSO) from the cells. The speed and duration of centrifugation should be adjusted based on the type of cells to minimize damage.

Why is it important to carefully aspirate the media after centrifugation?

Careful aspiration is crucial to avoid disturbing the cell pellet. The DMSO-rich media needs to be removed completely to prevent toxicity and ensure that the cells are healthy.

What should be considered when determining the suspension volume and flask size for the resuspended cells?

The suspension volume and flask size should be determined by the number of cells recovered. Seeding cells too sparsely can slow growth, while overcrowding can lead to cell death.

How can you assess cell viability after thawing?

Cell viability can be assessed by taking a small aliquot of the suspension and counting the cells using a hemocytometer or a cell counter. Viability can be further assessed using specific stains or tests.

What are some possible reasons for poor cell yield or growth after thawing?

Poor cell yield or growth could be due to issues such as low-quality frozen stock, improper freezing conditions, incorrect thawing procedures, or the use of poorly prepared or sterilized culture media.