While maintaining cancer cell lines originated from cattle squamous cell carcinoma we are troubleshooting an unusual problem as we are getting some spheroid like structures in our flasks maintained in DMEM-F12 with antibiotics. To exempt the probability of contamination we performed gram staining, Leishman's staining, and inoculation in both blood & SDA agar, and there was supposed to be no contamination. Then we performed immunostaining with oct-4 & TRA-1 which was available in our lab as a canine ADSC marker. We performed rt-qpcr with cattle specific primers for cancer stem cells, and it yielded that there is positive fold expression in these tumor spheres of oct-4, sox-2, lin28 & klf4. Then parent cancer cells and very high expression of CD24 did not match the product size when run on gel to confirm, but oct4, sox2, lin28 & klf4 matched their size. Then we tried to perform karyotyping from these Embryoid bodies (?) and got not exact spread but something like bundles of long chromosomes united at their centers and heavily condensed. We have done the soft agar colony forming assay, but still we are facing the problem that its growth is very fast and media color is towards yellow when kept in differentiation medium & HESC medium for a day. As our lab doesn't have a lab animal facility is there any way to know exactly what it is? RNA content is also low (9ug) and showing 200 bp difference in both 18s & 28s RNA size when run on bio-analyzer. Attached is a picture of what we are getting.
Be weary of your interpretation here, many cancer cell lines (or indeed immortalised cell lines) will, given the opportunity, round off the culture plastic and grow as spheroids. This may occur for a variety of reasons, probably the simplest being over-confluent monolayers (and given 'fresh' plastic cells that have lifted may wholly or partially reattach to the plastic). It is obviously difficult to infer exactly what might be going on from the information here. The work you have done to characterise the spheres does not reflect that you are making embryoid bodies, but most likely simply transcriptional changes to going in 3D space with differing cell contacts, nutrient availabilities and so forth.
Look back to your basic culturing conditions for reasons why the cells are rounding off, take out younger vials of cells, check your media, seeding densities, culture plastics, and so forth as there may be a simple reason for why your previously adherent cells are lifting.
thanks ANDREW HOLLINS; but if solely due to some culture condition problem it is occurring, then is it possible that these spheres are increasing in no. & making tumor like particle inside flasks while it is being maintained separately in different types of media. please suggest some way that can justify this problem. thanks again
I can't be sure from just looking at a picture, but I have to agree with the previous answer that your cultures look over-confluent. Maintain cultures no more than 75% confluency and see if the "spheres" go away. From the picture, those look more like clumps of debris to me than spheres, but I haven't worked with that specific cell line before.
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