Hello Deeptimayee Rout

There may be several reasons.

1. Mammalian cell lines can survive for up to one year when stored at -80°C. It is always better to store the cells in liquid nitrogen for long-term storage. Though the passage number is low, the period of storage at -80°C is beyond two years. The cryopreservation process may have compromised the cells' physiological integrity.

2. You should also consider environmental stress. Cells cultured under suboptimal conditions may not attach well. Factors such as

a. Contamination,

b. Fluctuations in incubator temperature,

c. Inappropriate cell culture surface/substrate,

d. Inappropriate environmental gas mixture,

e. The media may not be having enough serum/attachment factors.

can cause stress to the cells because of which cells may not be getting attached to the substratum.

You may improve upon cell attachment after thawing by using protein coated flask as well as optimizing the freezing and thawing protocols namely, by following the general rule of slow freezing and rapid thawing. If you plan to store cells for long-term storage, use liquid nitrogen instead of -80°C deep freezer to preserve cell viability.

Please note that poor cell attachment can lead to apoptosis and necrosis of unattached cells.

Best.