I am generating spheroids of squamous cancer cells and I see very clear spheroid generation within 48 hours of plating the cells in 60 well microwell plates.
When attempting to draw up the spheroid in a 200uL pipette tip, the spheroid degenerates. All other spheroids in that plate also disaggregate even without any disturbances. Has anyone else seen this issue or can anyone help me identify potential mistakes in my technique?
At 48hr, when cells are in hanging drop they get aggregated and at this stage they are not very compact. So, use a glass pipette (5/10ml)having wide opening along with a rubber bulb for drawing spheroids instead of 200ul pipette tip.
Usually I am growing spheroids in hanging drops for about 4-5 days before changing medium. Medium supplementation with a low concentration of methylcellulose also helps to form spheroids better.
Hello Nisitha.
It is not clear whether you used 60 micro-well plate (volume capacity 8-9ul/well). If so, the medium should have gone to acidic quickly. The cells may be damaged and dead after 48h. Check the medium pH by placing on white A4 sheet of paper along with freshly prepared dish to compare the color of the culture medium.
Deep round-bottomed 96-well plate (normal sized) full fluid capacity would help you to aggregates for further culture as hanging drops
Regards.
it was suggested if you use a plastic pipette tip, cut a bit from its head to make a wide opening and use it for draw up spheroids
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