Dear Scientist,
I am Looking for experts advice on handling SH-SY5Y (Neuroblastoma) human cell line
I am facing some practical issues in subculturing of the cells
After 5 days incubation in F12/DMEM (10% FBS) media most of the cells are floating, there are some attached cells too (limited numbers). But for my experiment I need attached cells.
Could you kindly share your experience how can i get more number of attached cells in T-25 Flask
Hi Murugesan,
The reason to pinpoint unattached adherent cells in culture is difficult as there are a lot of factors that might cause the inability of the cells to attach. Here are a few as per my experience:
- Contamination: This is the worst of all. It can be anywhere, in the media and reagents, inside the incubator, work environment or on the cells you are culturing. While some contamination are detectable under microscope or by looking at medium depletion (colour change) and turbidity, some unknown viral contamination are undetectable. However, if you got micoplasma contamination, which is highly common, you can know about it through some PCR or fluorescence based kits available. I recommend to check micoplasma contamination (because it is common) in your current batch or any batch that you will subculture later.
- Fluctuations in incubator temperature: Without appropriate temperature, your cells go under stressed condition. Better to check if your incubator have proper temperature maintained or if the sensors are giving the correct readings (troubleshoot).
- Insufficient or inappropriate cell culture surface/substrate: A year ago, I mistakenly cultured animal cells in bacterial culture dish and the cells did not attach. I hope it is not the case, but make sure you are using the proper culture surface.
- Environmental gas mixture is not appropriate: The CO2%, humidity are other minor but important factors.
If you try them all and find nothing or nothing works, you can try the followings:
1. Sterilise everything, use fresh materials and start a fresh batch of culture.
2. Treat the cultureware surfaces with extracellular matrix proteins such as collagen, fibronectin, laminin, or other factors to facilitate attachment. It can be the last resort as many adherent cell types will not require more than fit-for-purpose cell culture surfaces to successfully attach and form layers.
Hope this helps.
SHSY5Y when they are kept to long in culture tend to grow in suspension. It happens after ca 2 months of constant culture. Discard your cells, thaw new bank and start again. Sometimes after thawing I am using growth media with 20% of FBS to help them undergo stress connected with freezing. SHSY5Y cells are quite difficult to handle. Good luck!
SY5Y SH cells are rather fibroblastic cells that adhere to plastic. It is possible to have some of these cells that peel off to grow in suspension. But, in our hands, the cell layer remains stably anchored to the plastic. If all the cells take off, it is that you have an overdensity problem, but more certainly you have a mycoplasma infection, which I advise you to check.
Jean-Jacques Pin thank you for your suggestion. First time I'm hearing the word "mycoplasma infection" kindly explain in detail, do you have any protocol for mycoplasma infection assessment?
Mycoplasma infection is often provided by serum culture medium (FCS), or by trypsin used to detach cells. The first signs of infection are the detachment of the cell layer, followed by a premature yellowing of the culture medium while cell growth continues to decline. We use the Lonza Kit to detect the presence of mycoplasma. And we grow all our cells in the presence of Tiamulin, preventively because nontoxic, to avoid any external contamination. For curative treatment, ciprofloxacin is used at sub-toxic doses for the cell.