We are trying to establish culture of E0771 (murine breast cancer cell line) as 3D spheroids. Unlike other cells such as MC57G, E0771 cells don't form 3D spheroids on agarose bedding in 96 well plates.
Any suggestions are highly appreciated!
The cells are recently ordered from commercial vendor, I am not sure how to check if there is any cancer stem cells in the culture. Morphologically and even in orthothopic transplantation in mice they look normal.
Hi just you can try in ultra low attachment plate with proper growth factors (FGF, EGF etc.) in DMEM/F12 medium.
Hi, from your question, I am not quite sure, if you want to establish spheroids originating from one cell ("sphere formation assay" used for stem cells), or simply form a spheroid from multiple cells. for the latter purpose you could use either the simple "hanging drop method" (I have the citation somewhere, you can email me at [email protected]) or try http://www.microtissues.com/
in my hands (but I work with glioma cell lines) the 3D Petri Dish gives nice and regular spheroids in 2-3 days.
Thank you Petr! I just sent you an email.
Well, here I am trying to use around 5000 E0771 cells to make a 3D spheroid on 200ul medium on 50 ul of 1.5% agarose gel in 96 well plate. As you mentioned we are using pretty simple and well established method of soft agar colony forming assay but these cells never form spheroid in our hands. I am considering 3D petri dish that you mentioned, looks like its worth trying.
If you have any further suggestion/comments, please let me know.
Shahzad, I had the same problem with DU145 and PC3s. Agarose colony or spheroid formation assay is not suitable for all types of cells. I suggest you to use Matrigel mixed with agarose and try different combinations. There is another protocol for demonstrated on Neuronal stem cells in non adhering plates but Matrigel experiment would be the best additive for your stabilization. I hope this would help.
You can try using Hanging Drop Plates (www.3dbiomatrix.com) to assist spheroid formation. With those plates you can add a bit of matrix (matrigel or other), 2.5%, to your cultures to assist with spheroid formation. Another suggestion is to co-culture with a fibroblast. Matrix and / or co-cultures help cells that don't normally form spheroids (e.g., PC-3) grow as spheroids.
Thank you Sandeep and Meghan for your suggestions! I will try those and will get back to you with results.
As mentioned earlier, you could try the hanging drop method. It's the simplest. If you wish to continue following the liquid overlay technique (1.5 wt % agarose), you can employ a gyrator, so that the cells aggregate to the center concave region of the agarose coated well. It works well for me. People also try to microencapsulate them in alginate beads followed by poly l lysine and alginate treatment and use citrate buffer to liquefy the core to aggregate the cells. You could try this too.
I haven't worked with the cell lines you mentioned. So I'm not sure what to expect.
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