I have performed the primary cell cultures from Zebrafish Embryo 2-3 dpf using DMEM, DMEM+FBS 10%, and DMEM+FBS 10% with coated collagen dish. The results of cultures observed with inverted microscopy showed not very good results. Here are some related questions about it.
1. What might cause the cell is not attached (adherent)? Is contamination capable of causing non adherent cells?
2. What do you think about the images I have included? Can the contamination be determined from bacteria or certain organism? What cells or organisms are round in shape?
3. What are the factors interfere cell division and differentiation?
If you're thawing out a frozen culture, there always will be dead cells. They do look like dead cells. Change the media and see if you still have the same phenotype. If you still see the same phenotype, this would be the original cell population has a contaminant that you cannot get rid of. In this case, take out a fresh tube, thaw and regrow them.
Or if you're expanding from already growing cells, this could happen when you harvest the cells from the previous culture. If you aspirate cells up and down harshly, there would be debris. If you trypsinize at 37˚C, make sure they are completely detached until you spin at 300rpm for 5 minutes to remove the trypsin.
Hi Rovika,
1. What might cause the cell is not attached (adherent)? Is contamination capable of causing non adherent cells?
The contamination is indeed capable of causing non-adherent cells just because they are dead or dying. I would suggest to use new FBS (filtered) and new medium just to be sure that you don't have any other contaminations. Also, do you bleach your embryos before? That helps preventing contaminations. I also use 1/1000 of Pen-Strep the medium for zebrafish cells.
It also helps using a hood and incubator JUST for zebrafish cells.
2. What do you think about the images I have included? Can the contamination be determined from bacteria or certain organism? What cells or organisms are round in shape?
From the images I would say bacteria! The round shape are zebrafish cells (mostly dead) but between them you can see a big culture of bacteria. If you want to be sure if you cells are viable use the Methylene blue, and count the cells with Thoma cell counting chamber because the cells are so small that in Biorad or other counters it will not "screen" any cells due to their small size.
3. What are the factors interfere cell division and differentiation?
For this you may have to use some growth factors in the medium (like vegfa for endothelial cells, or FGF for fibroblasts) but this is something that I read, never tried it.
Hope I could help!
Good luck!
Thank you, that’s very kind of you Kyaw Myo Lwin , Shenglin Gao , Alexandra Veloso
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