Thaw 4 vials of N2102EP from stocks (All Mehrnoosh dated 4/2020)
1 vial - thaw into 1x T175 flask wih 35ml DMEM Glutamax + FBS
3 vials - thaw into 1x 250ml erlenmeyer flask with 100ml Freestyle 293 + FBS + 2mM L-Glutamine + phenol red
DAY 3 after thaw:
* adherent flask is ~50% confluent with lots of floating cells. Media changed
* suspension flask - ring of beached cells around the flask; after 3 attempts I could not get enough cells to count anything. Also do not see any dead cells or debris. Seed new 125ml flask with 15ml from main flask and 15ml new media. +15ml media to original flask
In suspension cells are growing in a thick clump in the center of the flask - one giant clump about the size of a euro coin. Under the microscope the clumps are significantly larger than HEK293TLD clumps, they are really uncountable.
There is a contamination problem. The adherent flasks became contaminated with rod-shaped bacteria, identifable by cloudy media the color of pink lemonade with a very strong unpleasant smell.
The suspension flasks have also had a contamination problem - when the cells are split, the new flask becomes contaminated within a week. I added Pen/Strep to the media to try and prevent this problem. We need to test our stocks.
FIRST HARVEST
SECOND HARVEST
Test IP First Harvest
Test IP Second Harvest
John thinks there is media trapped in the cell pellets because of the way these grow; if the pellets were not washed during harvest there is probably media trapped.