You should thaw frozen cells rapidly (< 1 minute ) in a 37oC water bath. Dilute the.....Remove the cryovial containing the frozen cells from liquid nitrogen storage and immediately place it into a 37oC water bath. Quickly thaw cells slowly, using pre-warmed growth medium. Plate the thawed cells at high density to optmize recovery. Always use proper aseptic technique and work in a laminar flow hood. Always wear personal protective equipment., including a face mase or goggles. Cryovials stored in liquid - phase present a rik of explosion when thawed.

Thank You, Sir. will try this process and let you know how it worked out.

Hi Naythan,

basically, always culture the cells at 2x10^5 to 1x10^6 cells/mL. However, as you say you do not even reach 10^5 cells/mL, I would suggest the following, in addition to Sylvesters remarks:

Thawing should be rapid (2 min). To increase initial cell density, try dispensing the thawed cells into a 25 cm^2 instead of a 75 cm^2 culture flask. ID-MEM cell culture medium may be better than RPMI-1640. Add 2mM Glutamine and 20% Foetal Bovine Serum (FBS) for cell culture.

It is important to avoid excessive alkalinity of the medium during recovery of the cells. Prior to addition of t the thawed cells, the culture vessel containing the complete growth medium should placed into the incubator for at least 15 min to allow the medium to reach its normal / optimum pH (7.0 to 7.6).

Wishing you best of success,

Friedhelm