Hello Mozhdeh Tafazoli

HFF-1 cells should not be semi-round 48 hours after thawing, but there could be a delay in spreading. While some stress after cryopreservation is expected, human fibroblasts are generally expected to attach and begin to spread out within 2–3 hours, with a significant increase in spreading by 24 hours.

To resolve this issue, you may want to consider the following.

1. Perform a trypan blue exclusion assay on a small sample of your cells. This will allow you to determine the number of viable cells and confirm if the semi-round morphology is due to a high proportion of dead cells. If the viability count is decent, give the cells more time to recover. HFF-1 cells may have experienced cryostress and would need more time to enter the exponential growth phase.

2. Gently replace the culture medium in your flask with fresh, pre-warmed medium to remove any residual DMSO and dead cells. This will also resupply the cells with fresh nutrients. The cryoprotective agent DMSO is toxic to cells, and prolonged exposure can cause stress and cell death.

3. Re-evaluate your thawing protocol. You should quickly thaw the cells without wasting much time, and handle the cells gently after resuspension as excessive centrifugation speed or rough pipetting can damage delicate cells, impacting their ability to adhere and spread.

4. Try to increase the cell seeding density next time, especially if the viability of your frozen stock is consistently low.

5. Re-visit your freezing protocol. Problems during cryopreservation, such as not following the technique of gradual freezing or insufficient cryoprotection, can reduce the overall viability of your cell stock.

6. Do not use cells of high passage number as they will be approaching senescence. Senescent fibroblasts have a reduced ability to grow and proliferate and will take longer to recover.

Good Luck!

Hi Mozhdeh Tafazoli

Using good-quality serum is highly important, along with all the points mentioned by Malcolm Nobre For HFFs, up to 20% FBS in thawing medium can be used. But remember to replace the media to your optimised concentration once the cells attain proper morphology.

Best of Luck!