Initially when we first started growing our SF9 cells in ESF 921 media, they were growing well. We were seeding at 0.5*10^6 to 1.0* 10^6 density using 60% fresh ESF 921 media (30.5 mL), 20% condition media (10mL), 15% FBS (7.5 mL), 10 microliter gentamycin and 50 microliter ampicillin in a total volume of 50 ML culture. With time, these SF9 cell growth rate decreased. However cell count increased from 0.5*10^6 to 2.5*10^6 within 4/5 days.
But after three cycle of seeding-growing-frozen stock preparation-again thawing and seeding, these SF9 cells are not growing anymore. I am seeding them at 0.5*10^6, 1.0* 10^6 and 1.5* 10^6 density in the same media combination, but they are not growing. After thawing frozen stock, all cells looks ok with a similar density as before, but they are not growing. The time difference between frozen stock preparation and thawing is around 15-30 days. and total 3 cycle of freezing thawing took around 3 months. Can anyone give me a proper instruction for long term storage and growing of SF9 cells.
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