3-D tumor culture - How to make spheroids? (Dec/05/2006 )

Hi there

Could you help me out with a completely new technique for me?
I have to produce tumor spheroids and I am looking for a simple method to do it.
I have heard of doing it in 24 well plates coated with agarose and somehow rotating it so that the cells will come together to the centre of the plate. Would you share your experience with me? It would be really, really helpfull!! Or do you have a paper that explains precisely how to do it?

Thanks in advance, a hug

Adri

Actually I am looking for spheroids made in agarose scafolds.
But thanks for the reference anyway!

Adri

Hanging Drop method

Trypsinize cells, centrifuge and resuspend in fresh medium such that you have approx. 2 x104 – 3,5 x 104 cells in 20 µl of medium. Pipet 20µl onto the cover of a Petri dish (of course the inside of it). Put 10 ml sterile water into the Petri dish so that the cells won’t dry out in the incubator. After 24-48 hrs you will obtain cell spheroids, which need to be transferred into 10 ml Medium in a 2% Agarose-coated Petri dish. These aggregates can be used in the collagen invasion assay.

Collagen Invasion Assay

Solution:
Cold Collagensolution (Vitrogen®3mg/ml, TypI) is mixed with cold 10x MEM to yield a final concentration of 2.4 mg/ml. If pH is not as cells like it add 0.1 M NaOH to adjust pH. Distribute solution onto 96-well-plate (100µl/well). Put 1-2 spheroids in each well. Put plates into incubator so that collagen polymerizes (30-60 min). After that add equal amounts of medium to each well. Cell invasion is observed for 1-5 days. If you want to treat the cells with substances, just add these to the medium which you put on top of each well.

[quote name='Jou' date='Dec 6 2006, 10:57 AM' post='79995']
Hanging Drop method

Trypsinize cells, centrifuge and resuspend in fresh medium such that you have approx. 2 x104 – 3,5 x 104 cells in 20 µl of medium. Pipet 20µl onto the cover of a Petri dish (of course the inside of it). Put 10 ml sterile water into the Petri dish so that the cells won’t dry out in the incubator. After 24-48 hrs you will obtain cell spheroids, which need to be transferred into 10 ml Medium in a 2% Agarose-coated Petri dish. These aggregates can be used in the collagen invasion assay.

Thanks for your protocol!
I have tried this week a different one. In a 24 well plate I put 200µl of an 2% Agarose solution and rotated it in such a manner that the agarose formed a sort of valley in the middle of the wells. Then I added the cells (100.000/well in 300µl medium) and left it in the incubator for 30'. finally I took the plates and made circles over the surface of a table (more or less 1mx1m) for 5'. I got nice rounded spheroids. But anyway I will try your protocol, because rotatig the plates myself for 5' to 10' (depending on the size of the cells) is not a funny job :-)
have a nice weekend,

Adri