LaCava Research Wiki

23 April

After meeting with Margit from ROME and Samira about the drug assay, we know we need to leave the drug on the cells for at least 72 hours to alllow for 2 complete cell doublings.

N2102EP in our hands typically needs a media change every 2-3 days, so this means using our standard protocol would require a media change of 48 plates while the drug is on the cells. Increasing the individual flask volumes (while not exceeding the manufacturers recommended max volume) could help avoid this media change, if it allows the cells to remain healthy for an additional 1-2 days without a media change.

I designed this small pilot project to quickly assess whether this strategy might work.

Thermo Nunc Flasks Volume Recommendation

Corning Flasks Volume Recommendation

Thaw two vials N2102EP from cryostorage, one to be treated with standard protocol (CTL = 10ml media in T75 flask) and one with increased media volume (+M = 15ml media in T75 flask) to see if media increase corresponds to longer time between necessary media changes. (Used cell stocks 44 & 45)

• Thaw vials in 37C water bath.
• Prepare 2x 15ml conical tubes with 10ml media each
• Resuspend cells in the conical tubes (inside biosafety cabinet)
• Spin cells 5 minutes @ 1000g RT
• Remove supernatant with pipette (vacuum may dislodge pellet)
• Resuspend pellet in 5ml media
• Add to prepared flasks
• Distribute cells throughout flask w/side-to-side movements
• Place cells in 37C incubator, 8% CO2

25 April

2nd day since cell seeding CTL flask is ~ 50% confluent, media starting to turn orange +M fllask is also ~50% confluent, media is still mostly red.

26 April

3rd day since seeding Split cells to new flask with accurate cell counts for ideal comparisson

CTL flask has 2.9 million cells/ml +M flask has 3.2 million cells/ml Since seeding density was not calibrated, the difference in cell numbers are not notable. Media in both flasks is orange, but CTL flask has significantly more floating cells and media is beginning to turn yellow

Seed two new T175 flasks from the +M flask (cells were less stressed so new round is only these cells for "fair" comparison Each T175 fllask was seeded with 2 million cells. CTL flask has 25ml media (LaCava Lab standard) and the +M flask has 35ml media.

30 April

Both CTL and +M flasks are orange, but +M flask has only just started to turn orange on the 5th day. So at this cell density, icreasing the media volume decreases the need for media changes between cell splittings. Need to calculate which strategy is more cost effective - probably increasing media volume in most cases.

Samira to perform one more time while I am away from the lab. She will seed 2x T175 flasks with 4 million cells each to test this theory with a higher cell density.