As I know SNL76/7 is a passage-independent feeder, hence I assume they will not change morphologically and karyotypically, after indefinite sub-passaging. However, recently I noted the changes in one of the flasks (attached photos below), there are 2 morphologically different cells appearing. I wonder if anyone has encountered this before, or if anyone can share more about SNL 76/7 culture experience and tips. I am totally new to this culture and am using this feeder to grow iPSC. There are more questions regarding iPSC growing on this SNL feeder which I have posted as different questions. Kindly advise and your comments are highly appreciated. Thank you.
I have worked on several fibroblast cell lines and stem cells. In my opinion this may be a very minor issue. My hypothesis is that the morphology has transiently changed due to some kind of stress. I would passage the fibroblast one or two (48 hours culture duration) times in fresh flasks and check if the morphology change still occurs.
Biswajoy Ghosh Noted for your advice. I guess the fibroblasts are not senescent right? As the senescent cells should be very large and having a very flat cytoplasm. Thank you.
Hi Teo, what is the passage number of the cells?
Stresses do induce scenescence, but transient morphology changes, vescicle formation do occur and after a couple rounds of healthy passaging it can be fixed sometimes.
If the issue still continues you can check for trypan blue viability, cell proliferation markers like ki67, and DNA damage markers. DAPI can reveal some vital clues on DNA damage. Nucleoli number increases. The Senescence associated beta galactosidase assay need to be performed to confirm. Even a simple high mag phase contrast for nuclear features can be helpful. You do need to compare it with a low passage cell.
Teo Ting Ting Yes, it is. However, I was thinking on the lines that passage independent cells won't undergo a plastic change in morphology with the higher passage numbers, but can be affected by senescence or stress.
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