Hi Geraldine, V- or U-bottomed wells will work better. If you use other blocking agents than agarose such as BSA, you need to use untreated-polystyrene plates.

Thanks for the input Kiran, the V or round bottomed wells would work better, but i was just surprised to find published protocols stating that the same could be done with the flat bottomed plates. Just a thought.

Hmm... I wonder if with sufficient agarose volume, you can form a meniscus that would change the curvature of the flat-bottom sufficiently to effectively form a U-bottomed dish. Atleast sufficiently to increase the encounter likelihood between cells.

Yup, you are right. The meniscus would help to produce a concave surface for cells to come together, however it is less efficient than a true round bottomed plate. Thanks for the input though!

I have done this recently. It is as efficient as U-bottomed plate. But if you have U-bottom plate, it is not necessary to perform the coating in the flat-bottom plate.

Could you share with me how you did it, Weihua?

Here is our protocol:

1 Disolved 0.2g agarose in 20mL sterile distilled water in centrifuge tube, heat to 90 degree for 30min.

2 Transfer the 1% agarose solution to laminar bench. Keep it warm all the time.

3 Cover a 96-well flat-bottom plate with 1% agarose, 100 uL each well. Plates are allowed to solidify at room temperature for at least 2 hours.

4 Seed the suspending cell as usual.

Good luck!

I did some experiments with spheroid assay according to Nature Protocols 4, 309 - 324 (2009). http://www.nature.com/nprot/journal/v4/n3/full/nprot.2008.226.html. I did in some breast cancer cell lines. In my hand, mcf-7, BT-474 and HCC-1954 cell lines were easily formed a single spheroid. I think cell number and FCS concentration are the most critical points. 

Corning have a new Spheroid plate, 96 well (also 384 well), ultra-low attachment surface, optically clear round bottom with black opaque microplate body.  Allows for growth and imaging of a single spheroid per well in the same plate. Product #4515

http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?categoryname=&productid=4515(Lifesciences)

Hello

I have done the same as you Geraldine and I obtained a single spheroid/well. However the problem is when I try to remove the spheroids from the well. It has tendency to desintegrate the spheroid. Any idea how to remove it from the well?

Thanks in advance

How long do you keep the spheroids in culture? In my personal experience they become more solid the longer you keep them in culture and then they are easier to remove. Just use a pipette and be careful.

Has anybody  tried the agarose-coating to obtain mammosphere from cell lines, such as MCF-7 or MDA-231 or MDA-468?

Thanks for sharing. Will the spheroid be strong enough for cell seeding in a spinner flask bioreactor after surface modification.

Hello every body, did anyone created spheroids from U-87 cell line? What is the suitable percentage of Agarose to be used? How long should I keep them in the Incubator in order to obtain a single spheroid/well? regarding cell seeding, is it recommended to load the cells inside the gel after it becomes solid? Or should I load them as usual? in this case the cells will remain above the Agarose. Really appreciated your help

Shada,

We use breast cancer cell lines and have been getting pretty nice results with 1% agarose. You plate the agarose and then allow it to completely solidify overnight. After that is when you add cell suspension. Additionally, you should spin them in a hanging bucket centrifuge at 1000g for at least 10 minutes so they form spheroids. As to removal of spheroids from the wells, we've found that using a Pasteur pipette works really well.

Theodore I tried spinning the cells at 1000g and 300g for 5 min and 10 min but the cells did not form any spheres, they remain as flat irregular flacks. I tried loading the cells suspended in a given volume of medium (100ul and 200ul) as well as loading a concentrated cell suspension just in each well and still have the same problem. what other options can i try to get my cells adhere to each other and form spheres??

Also using hanging droplet method can be a good alternative to culture cells in spheroid form

Hello everyone. Did anyone manage to create spheroids using 1% Agarose coating on the HUH7 cell line? Are there any special considerations that should be taken compared to the previously mentioned cell lines?

In the protocol, they sterilized the agarose by passing it through a 0.2 mm filter. but this was not possible. it freezes directly. Will it be sterile enough if I autoclave it? Also, do you think it will be toxic in cell culture? Does the type of agarose matter?

Belma Nalbant in my case, I sterilized 1.5% agarose solution at 121 Celsius, 10 minutes in autoclave. And it is not toxic for my cell line.