Hi everyone,
I am new to cell culture and I am trying to maintain HEK 293 cells. The cells are 100% confluent and so my supervisor told me to passage , expand and freeze them as I want. I passaged them into a new flask but I want to know when is the good time to expand and also freeze them?
Thanks!
Dear Keerti,
we used to passage cell cultures every third day (Monday and Thursday), no earlier and they have to be in 100% monolayer. If you do not want to expand them it is ok passage them once a week.
For freezing, I would use cells from three day culture.
Good luck!
Marcela
Hello Keerti,
HEK-293 is a cell line that grows well. Subcultivation Ratio is 1:6 to 1:10 weekly. Medium Renewal: Every 2 to 3 days. If you need cells for experiments or to expand them, you can split them twice a week (Monday and Thursday), as mentioned above. 24h before freezing, growth media should be replaced with fresh one. So, you could split the cells on Monday, feed them with fresh media on Wednesday and finally freeze them on Thursday.
HEK 293 look big and expand rapidly depending upon the quality of cell lines used. I think the passage of the cells depend on the growth pattern whether they are grew in T25 or T 75 flask.
Also the confluent condition decided whether the cells ready for further sub culture.
Hi Keerti
HEK cells are very fast growing cells. Anything between 1:6 and 1:10 splitting is fine depending on your experiment; media change in alternate days. Its always better to split any cells before 100 % confluency. You can freeze two to three vials from a confluent T25 flask and can later revive one in a T25.
Thank you everyone. I feel comfortable doing expansion and freezing cells.
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