Hello everyone.
I am looking for suggestions for HL60 cell line culture. Our lab is new to suspension cell culture. we bought the cell line from National Center for Cell Sciences (NCCS) Pune. Upon arrival, the cell was absolutely fine, having a doubling time of around 48 hours and we were able to freeze 10 vials with 10^6 cells/ml (90% FBS + 10% DMSO). But after 6 months in liquid Nitrogen when we started culturing from those vials it seems that the cells are not proliferating. We are seeing cell clumps forming and after 3-4 days all of these cells are dying (when checked using trypan blue). Can you help me with this problem? We are unable to point out the exact cause of the situation.
Thanks
Hello Samya Dey
When the cells first arrived in the lab from NCCS, they must have been in the transport medium, I suppose. I do not know how did you further process the cells. If I were you, I would have first wipe the outside of the flask with 70% alcohol to avoid contamination, and kept the flask in the CO2 incubator at 37 degree C as the cells would have been in stress during transit. The very next day I would centrifuge the suspension cells at 700-1000 rpm for 10 mins and resuspended the cells in fresh growth medium, and cultured the cells for a few days observing the heath status microscopically. I would then subculture the cells (healthy cells) on a larger scale making many flasks for the purpose of freezing at 1 million cells per vial. Freezing of cells have to be done gradually by reducing the temperature at approximately 1°C per minute using a controlled rate cryo-freezer or a cryo-freezing container such as “Mr. Frosty.”
Thawing of cells have to be done rapidly by placing the freezing vial in the water bath at 37 degree C. Immediately transfer the cell suspension to a large volume of pre-warmed cell growth medium (~10 mL/1-mL aliquot of cells), and centrifuge the cell suspension at 800-1000 rpm for 5 mins and resuspend the cell pellet in fresh growth medium. Culture the thawed cells in T-25 flask. About 24 hours after thawing, you can observe your cells under the microscope to make sure they are healthy and behaving normally.
Proper freezing and thawing procedures need to be followed to maintain cell viability.
Hope this information will help.
Good Luck.
Hi Samya,
It seem some problem ccurred during freezing or thawing procedures. Did you perform a slow freeze firstly -80 ºC/ 24 h (min 24 h, max 72 h) before passing to N2? And regarding thaw, did you put the cryotubes on ice just when you take them out of the freezer before put into a bath at 37 ºC/ mild shaking?
When we cultured HL60 cells, we grew them in suspension culture in RPMI 1640 medium supplemented with 10% FBS in air/5% CO2 at 37 ◦C. The cells were sub-cultured as required every 2–3 days. We froze these cells by resuspending a soft cell pellet in the same media but containing 10% DMSO. Then sealed vial of this mixture were first cooled relatively slowly at about 1◦C/min, in a bath of CO2 acetone, to around -80◦C, before being transferred to liquid nitrogen. Vials were thawed after storage in a water ice bath, prior to addition to RPMI 1640 medium supplemented with 10% FBS in air/5% CO2 at 37 ◦C. It also may be wise to gently wash the cells in RPMI 1640 medium plus 10% FBS, as DMSO can induce differentiation in leukemia cell lines.
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