Could it be that you have some fibroblasts deriving from spleen? This is quite common in my experience, after 8-10 days from fusion they may form adherent clusters but are completely harmless to hybridomas

Thanks for the response. They don't have fibroblast morphology i.e. they aren't elongated, in the tissue culture-treated flasks that I'm using (I'm aware that I don't need to use tissue culture-treated flasks to grow hybridomas, but that's all we had in the lab). Or is it possible for splenic fibroblasts to look spherical?

This often happens to me - I think it's a product of using serum containing media (if I transfer to serum free I have no problem). I haven't noticed any variation in antibody production. I tend to use a cell scraper when expanding, but other than that treat them the same.

Hi Daniel

Don't worry, in my experience, certainly, some clones would have slight adherence to the culture flask, especially in the presence of fetal bovine serum.

To be sure that you have a clone, you may perform several limiting dilution series.

Test the expression of each obtained clone, and choose the best for your purpose.

I also agree with Aida - some hybridoma cell lines just grow that way.

Large scale cultures of hybridoma cells (>1-100 L) are often carried out in suspensions under continuous stirring (to improve O2 transfer) and the cells are often sticked on beads of polysaccharide to minimize shear stress. Many clones of hybridoma grow well when fixed on a support and continue to produce immunoglobulins .

Data in the literature suggest that exosomes mediate attachment.  If FBS is necessary for your culture and you would like to avoid adhesion, you may wish to deplete the FBS of exosomes by ultracentrifugation

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0024234

http://www.fasebj.org/content/early/2004/06/02/fj.03-1094fje.long

I didn´t generate hybridoma cell line myself but I have experience with some suspension and hydridoma cell lines. Some of them just like to attache to the flask and also some are more like a single cell suspension whereas for other lines the cells tend to stick together. I turn the flask upside down, so that the non-treated surface is in contact with the cell suspension and I put the top on a spare lid, so that the flask has an angle. If the volume is not so big, it also works fine to have the flask vertical instead of laying them flat. As long as the morphology of the cells, which are attached, aren´t change, everything sould be fine. But I would be carfeful if they change the shape.

I too am having a problem with hybridoma cell lines adhering tightly to T75 tissue culture flasks.  The CDC Hybridoma Core did the fusions and I got the clones in 96 well plates, ready to screen supernatants by ELISA.  Clones were subcultured to 24 well plates, to 6 well plates, and finally to T75 flasks.  Our media is  IMDM +5% low IgG FCS + antibiotics +glutamine. Core personnel use the same brand T75s and media as me, the only difference being the peptide immunogens used to generate the splenocytes and B cells.  I am going to try using non-TC-treated plasticware and see if that solves the adherence problem. We are using cell scrapers to harvest hybridomas from T75s which is very time-consuming and not ideal for the cells. 

It may be related to the myeloma cell line used during fusion. SP2/0-derived hybridomas tend to attach more than NS1-derived hybridomas. That said, I also have observed different growing pattern hybridomas in the same well. I believe this variety on the same well is related to random gene loss during the early hours after cell fusion. Remember that after fusion, quite a lot happens, changing the hybridoma phenotype.

You have to test the production of Antibody by the various clones, some people are growing poorly adherent hybridoma cells in  0.5 or 1 l vessels under continuous stirring thus improving O2 supply. You should also be aware  that Ab production even by a single clone is not constant and may vary along the life of the culture. 

I have this problem too!!