10X solution:

For example, dissolve 2.4 grams of polyhema in 20mL of 95% ethanol (in a 50mL falcon tube). Then, seal it with parafilm, and put on a wheel at 55 Celsius for an overnight. This solution is 10X. Dilute it 1:10 in 95% ethanol to get the final solution at 1.2%.

1X solution:

If you want you can also directly prepare 1x solution by dissolving 12g polyhema in 1L of 95% ethanol, overnight at 60C with a stirrer in agitation.

Then Filter the solution with a 0.2uM filter unit before using it.

Prepare the plates under a sterile hood. Let the ethanol evaporate completely (leaving the plates without the lid under the hood for an overnight), repeat the procedure (e.g. next morning), wait other 24 hours (2nd evaporation time) and finally store the plates at 4C. Note that evaporation time depends on the plates you are using, can be less than stated.

No further sterilization is needed

Federica you can refer to these publications of my group: search for >>> Tosoni D, Pece S, Di Fiore PP, there is also a very recent review: Functional purification of human and mouse mammary stem cells.

10X solution:

For example, dissolve 2.4 grams of polyhema in 20mL of 95% ethanol (in a 50mL falcon tube). Then, seal it with parafilm, and put on a wheel at 55 Celsius for an overnight. This solution is 10X. Dilute it 1:10 in 95% ethanol to get the final solution at 1.2%.

1X solution:

If you want you can also directly prepare 1x solution by dissolving 12g polyhema in 1L of 95% ethanol, overnight at 60C with a stirrer in agitation.

Then Filter the solution with a 0.2uM filter unit before using it.

Prepare the plates under a sterile hood. Let the ethanol evaporate completely (leaving the plates without the lid under the hood for an overnight), repeat the procedure (e.g. next morning), wait other 24 hours (2nd evaporation time) and finally store the plates at 4C. Note that evaporation time depends on the plates you are using, can be less than stated.

No further sterilization is needed

Federica you can refer to these publications of my group: search for >>> Tosoni D, Pece S, Di Fiore PP, there is also a very recent review: Functional purification of human and mouse mammary stem cells.

If you are using polyhema to prevent cell adhesion and make spheroids, you might also like to consider round-bottomed ultra low attachment 96 well plates from Corning and increase the density of the medium slightly using carboxymethylcellulose to obtain highly reproducible round spheroids....if interested in this alternative method let me know.

Yes, ultra low plates are also a good option. The trade off is the price (3-times or 4-times higher).

Thanks a lot Matteo for your advices! Maybe I made some mistakes with time and temperature to dissolve Poly-HEMA. I will try to do your protocol!

Jorge, according to your experience, is it better to use round-bottomed or flat-bottomed ultra low 96 well? I want to try 96 well to facilitate automated sphere counting in the future...

Thanks Jorge, I use already the flat-bottomed 96 well plates with agarose to make spheroids and now I'm curious to try poly-HEMA. Do you have experience with round-bottomed 96 well plates?

Hell, Mr.Jorge Burns i have seen your reply here and its interesting can you please elaborate spheroid formation assay by using ultra low attachment plate along with carboxymethylcellulose.

Hullo Matteo and Federica, we have explored round-botted, flat-botted and also V-shaped wells. A direct comparison suggested that shape was very significant and round-bottomed was the best option. You might like to see my Tissue Engineering 2010 manuscript, which shows multicellular 3D spheroids made this way.

Dear all, I would like to try to use the U shaped 96 wells plates coating them with agarose and polyhema. Have you tried the hanging drop technique? I started with it but actually it is quite time consuming and I have some problems to recover my spheroids.

Simona, Corning manufacturers plates with an ultra low attachment coating that do not require coating with polyhema. They are the best non-attaching plates I have tried, so much so that I suggested they should make 96-well U tube plates with the same coating. They did and they are broadly available #7007. They work very well, especially if one uses methyl cellulose to increase the density of the medium slightly thus improving spheroid reproducibility. Hanging drop plates are also on the market (http://www.selectscience.net/SelectScience-TV/Videos/3d-cell-culture-using-perfecta3d-hanging-drop-plates-from-3d-biomatrix/?videoID=1337) and you would do well to compare the two methods, it could form the basis for a brief technical paper.

thanks Jorge,

i have just ordered the perfecta3D plates. I will try soon. thanks for the Corning reference. I have now a look

Jorge Burns, i tried methyl cellulose (Sigma M0262), not carboxylmethylcellulose. How much of a difference is there between these two? I made a 1% and a 2% solution and diluted 1:1 with 2x media fro 0.5-1% final. I noticed that there were fine crystals in the media and I think it interfered with the sphere formation. I see that Sigma also has carboxylMC. Which viscosity should I try low, med or high? I would like to filter the media and retain the spheres for subsequent passage. Can carboxyMC be filtered?

Dear Matteo,

why did you chose to use 1.2% polyhema coating instead of the common 0.5%?

How many µl well do you use?

thanks!

This article might help

http://www.jcancer.org/v02p0507.htm

J Cancer 2011; 2:507-514. doi:10.7150/jca.2.507

Article Rapid Generation of In Vitro Multicellular Spheroids for the...

I realize my previous answer really didn't help.

I found this protocol awhile back. If you can't get access to this publication let me know I will email it to you.

Cheers,

http://www.ncbi.nlm.nih.gov/pubmed/22914933

Mary Goldring describes the method in detail in Chapter 5 of "Methods in Molecular Medicine" Human Cell Culture Protocols, edited by Joanna Picot, Humana Press. Page 73.

Federica, please find attached the protocol

Ciao