Recently, I had a below comment from a journal that asked me to do a spheroid cuture without hydrogels or matrigels. As far as I know these materials are necessary, so I tried to find the new method that the rewviewer1 suggested, however I failed. I am carefully asking your advice.
Reviewer 1: While the responses to most of the queries makes sense, the one about growth in ultra low attachment plates inducing anoikis and therefore studies cannot be performed to identify spheroid formation is not correct. It appears that the ULA plates they have used in this manuscript has a hydrogel of some sort that does not allow attachment (and this was not mentioned in the original write up). There needs to be details of the unique hydrogel used in the current study because lots of studies have demonstrated that hydrogels are excellent scaffolds for mechanical support while directing cell adhesion, proliferation, differentiation, morphology, and gene expression. Furthermore, hydrogels are also used widely for 3D cell cultures. A second point that is important to consider is growing cell in ultra-low culture conditions that allow spheroid formation. This culture system does not need to employ any hydrogels or Matrigel.
Hi Soo Young Jun
If you tell us the type of cells which you were growing on hydrogel or matrigel, and the purpose of the study, then I think members can give better answers.
'I do understand that your manuscript might in the review or you will be going to communicate further' but more general details if you are providing then it will better.
My reply is based on preliminary understanding,
-For spheroid culture for cancer cells is possible WITHOUT any substratum, using the ultralow adherent plate and media with a specific component which enriches spheroid then there it is possible. Cancer stem cells culture as spheroids.
-Other forms of spheroid culture could be a hanging drop method where you don't need any support like embryonic stem cells are used to grow. Well, culturing needs very specific cell numbers and specific media.
But all my answers will depend on your cell type followed with purpose.
Regards
Saurabh
I would highlight the fact that the used hydrogel not the perfect type for this cells and highlighting the possible functional groups that help such cells for attachment.
Hi Soo Young Jun ,
I agree with the other comments that more detail about the cells you are using would be helpful in answering your question.
Like Saurabh mentioned, hanging drop is a good method for spheroids if the reviewers do not like your current method. Cancer cells and mesenchymal cells usually form spheroids quite easily using hanging drop or ULA plates. Not all cells can form good spheroids, however. For example, I routinely work with fibroblasts and HUVECs in ULA plates. The fibroblasts form very good, mechanically robust spheroids. The HUVECs do form "clusters" but they are not nearly as mechanically robust because they want to be surface cells, and putting them into a spheroid is an unfamiliar environment for them.
Have you been using a hydrogel to support your cells in this work?
I am wondering if the reviewer is confused, because they refer to a anti-fouling hydrogel on the ULA plate surface, but also talk about hydrogels being good for cell adhesion. If you are using a ULA plate, these are coated with materials that make a very thin coating that is often referred to by the manufacturers as a "hydrogel". These "hydrogels" are supposed to be non-fouling, which is quite different than Matrigel, gelatin, or other cell culture hydrogels derived from the extracellular matrix that cells usually bind quite easily. Without more information, it sounds like the reviewer may be conflating these different hydrogel types.
Thanks,
Caleb
This can be done using the hanging drop method. See Article A Simple Hanging Drop Cell Culture Protocol for Generation o...
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