which plates are you using? (treated) U bottom or flat bottom with agarose?

Hi Amit, we have managed it quite successfully using the following method but it will depend on how you have formed your tumourspheres and whether they are a true tumoursphere (spheroid) or just an aggregate of cells.

We use 96 well ultra-low attachment round bottomed plates from Corning (code 7007, they are much cheaper thean ones from certain other companies), plate the cells in 160ul of their normal culture media (the number of cells to seed is dependent on the cell line and size of spheroid you want - you should determine this experimentally), centrifuge at 1000 x rpm for 3 mins and carefully and gently place in an incubator to allow the cells to form into a spheroid for 3 days. This method should generate a single spheroid per well.

After 3 days incubation, compounds can be added to the spheroids. We do this by preparing a 5x final concentration in normal culture media (max DMSO 2.5%) and then very gently adding 40ul per well of spheroids. However, if your cells have formed a true spheroid, they should be quite robust by this point. Alternatively, if you want to see if the compounds have an effect on spheroid formation, they can be added immediately before the centrifugation step. At the desired time point, spheroid viability can be determined using e.g. cell titer glo, imaged etc etc.

This method has worked quite well for HCT116, HT29, DLD1 (see Tabusa et al, Mol Cancer Res 2012; Rawlinson & Massey BMC Cancer 2014) and other cell lines.

Hope this of help

Andy

Thank you Andrew J Masssey and Emanuel Kreidl. Actually I want to use flat bottom plate and not the round bottom plate without agarose.

Hi Amit

The first concern, as said above, is to assess whether or not a true spheroid is formed, and not just a cluster of cells.  Spheroids should withstand gentle addition of drug-containing media.  If the cells are easily dislodged from the  cluster, then you should consider using another method, such as the overlay method.  You can first culture the cells to spheroids in a small volume and then add a soft Agar overlay, which should minimize disturbance of the cluster on addition of drug-containing media.

Best wishes.

Celia Snyman