Hello Sayani Das,

I have experience in culturing the primary shrimp cells collected from some organs, such as the heart, muscle, and androgenic glands. I had the same issues with debris. In my opinion, there are some potential reasons.

1- The contamination of mycoplasma. If cells were isolated from fish gonads (primary cells), it was very easy to get mycoplasma infection. PBS or HBSS cannot help you solve this problem.

=> You can use the mycoplasma test kit to check. If there is a positive result with mycoplasma infection, you can use a Mycoplasma Removal Agent to add to the cell culture medium. The drawback is the cell growth rate might be reduced.

BTW, to reduce the contamination, you should sterilize your tools carefully before experimenting, wash the organs many times in the PBS + antibiotics, and culture the isolated cells in the cell culture medium + antibiotics.

2- I'm unclear whether your cells are primary or cell lines. If they are primary cells, they will easily die after several days of culture because they are not stem cells. The debris might be from this reason.

3- It might be that the cell culture medium is unsuitable for this cell type, so the cells might have died. You should be careful when checking the cell culture medium's salinity, pH, and osmotic condition. Your cells are fish cells, these conditions will affect a lot on the cell health.

Hope that my experience is helpful to you.