Hello Kimmie B. Christensen

After thawing SUPT-1, the cell viability is likely to drop to less than 50%. However, the cells recover during the following 5-6 days. The process of thawing cells should be done quickly. Your thawing process seems to be okay.

You need to consider other factors as below.

Before freezing, please check the health of the cells. It is important to perform a viability count using Trypan Blue. Check for contamination via sterility evaluation and mycoplasma testing. If you refresh the growth media 1–2 days before freezing, it will ensure the cells are healthy and in an active phase of growth. Cells should ideally be at around 70–80% confluency upon harvest for freezing. The concentration at which cells are frozen may vary between cultures but it is typically in the region of 1 x 10^6 – 5 x 10^6 cells/mL in freezing media. Freezing cells at too low or too high of a density can impact viability and should be avoided. I would not know the ideal cell concentration for freezing SUPT-1 cells. Usually, 2 x 10^6 cells/ml are frozen per vial.

How was the freezing process carried out?

To maintain viability, the freezing process should begin as soon as the freezing medium has been introduced to the cells. Placing the cryovials on wet ice before transferring them to the freezing container can expedite this. Freezing cells slowly is essential to prevent intracellular ice formation, and this may be achieved using a freezing container that provides a freezing rate of 1°C/minute. The cryovials should be placed at -80°C for a minimum of 4 hours but ideally overnight. Once cells have been frozen at -80°C, you may transfer the cryovials to liquid nitrogen. Do not leave the cells at -80°C for long periods as this can affect cell health.

Hope this will help!

Best.