I am trying to isolate single cell colonies from breast cancer cell lines. After isolating , I let them grow in 96 well plates. Initially for a week they are fine. But as days pass, the colonies seem to become unhealthy and necrotic. How much ever I wash them with 1X PBS and change media and supplement with FBS , they do not show a healthy morphology. How do I ovecome this.
Thanks in advance.
Hi Vandhana, my colleague also did this for colonies isolated from nasopharyngeal carcinoma cell lines based on differences of morphology. He just leave the cells in 96well with media wihout changing the media too regular as cells released growth factors. Changing the media regularly will wash away these important growth factors. Try not to change the media regularly or maintain 50:50 fresh media:old media, once a week maybe or even 2 weeks once (make it 200ul/well so that the media will last longer without the need to change fresh media). It will take weeks for the colony to confluent. My colleague managed to grow 3 different NPCs colonies into T25 now from 96 well plate. Hope this will help.
Reagan is correct. You should not be washing young colonies, but rather supplementing partial fresh medium onto the already-conditioned medium.
You may also try to use conditioned medium.
A more detailed description can be found here on RG:
https://www.researchgate.net/post/Does_anyone_have_an_idea_how_long_it_will_take_for_a_single_mcf7_cell_to_grow_into_a_clump_or_double_for_that_matter
Did you ever take in consideration that your cells may not be able to survive when they are single in 96 wells? In the past I tired to do this also with our cells (HCC cells) and it wasn't possible to grow them like this. Even with conditioned medium, it was not possible to grow them alone. Instead of seeding them on 96 well plates, you can also try to seed them in a very high dilution on 10cm plates and wait until colonies are forming, then you can pick them and transfer to 96 wells!
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