Hi all,
I purchased the SH-SY5Y cell line from ATCC (CRL2266). When I try to grow them in DMEM:F12 (1:1) media, the cells grow well for 3 days and then completely detach. I have also observed the same behavior in DMEM only.
Can anybody shed light on this behavior, and give me some tips to prevent this?
Thanks,
Subramaniyam
The cell line SH-SY5Y are semi adherent cell lines, meaning that they have the property to grow as adherent cultures as well as a suspension culture.
I would suggest adding Bovine serum albumin to the flask to improve adhesion.
When I checked the cells during the first three days there were very few floating cells. I am quite surprised that almost 100% of the cells detach within a day! I will try the BSA addition, though.
Here are recommended conditions for cultivation: https://www.lgcstandards-atcc.org/Products/All/CRL-2266.aspx?geo_country=sk#culturemethod
cultivation medium should be DMEM:F12 1:1 + 10% FBS (from my experience SH-SY5Y did not grow in DMEM as nicely as in DMEM:F12)
and some tips:
- before you add cells, place the cultivation medium in cultivation flask for approx. 20 min to cultivation box (let it saturate with CO2 to adjust pH)
- thaw cells in water bath as fast as possible
- wash cells in warm cultivation medium and centrifuge them to discard the resudues from cryopreservsation medium
- and then resuspend the pelet and add it to cultivation flask with CO2 saturated medium
good luck!
I added 10% FBS and 1% penicillin to the DMEM:F12 and to DMEM only.
Hi, I would suggest that its depends on your experiment, in which the cell lines to be used. So it ll be fine that if we chose a type of cell line according to our kind of investigation or assays.
You have mentioned that after 3 days its getting detached, but for what purpose you have chosen this cells?
The growth nature of SH-SY5Y cell lines is itself like that - as Mr. Shiv kumar mentioned it has both adherent and non-adherent base of growth characteristics.
some more tips: choose different cell lines which is obligate adherent type (Eg: SK-N-SH (ATCC® HTB-11 )
If the cell lines is really adherent type, still you are facing kind of issues, then you have to consider... culture flask-material made up of, Temp and pH of medium while sub-culturing, CO2 concentration in incubator, would be better if you change medium daily if the medium colour has changed, ultimately sterility of lab and materials
All the best....
Hi Ravichandran, SH-SY5Y have both adherent as well as suspension growth nature. But as you had mention that your cells were completely detached after 3days, Did you check whether they are growing in clumps. if so, then your cell are normally growing. Please do not get panic.
Hi Vinay,
No, they are not growing in clumps. One observation I made was that there seems to be a lot of debris post the 3rd day when the cells are floating.
Best regards,
Ravi
ok.... Did you check for the contamination. If not, check that their are contaminated with bacterial infection and also check for their media color. if possible for you, could you share the pic of flask under the microscope.
Hello, I do not know if you still have doubts, but I started working with this lineage a few months ago and what I realized was that it takes even days to establish. The first time I got it, I left 5 days with the same medium (DMEM / F12 + 10% BFS) and they don't adhere very much or those that were adherent don't grow. Then, after 5 days I passed this medium from flask to a tube falcon, centrifuged and added half new and put back to the same bottle from where I had taken initially. After that, in 2 days they began to adhere better and grow.
Another tip is to keep in small flasks (25 cm2) until reaching confluence.
Hi Ana Paula,
I'm using both 75cm2 and 25cm2 culture flasks, and I noticed that they grow faster in 75cm2 flasks. When cultured in T25, they keep growing in clumps, but don't form a monolayer (and the medium is depleting very slowly-which means that they are in deed growing more slowly?). However, when cultured in T75, they adhere much easily and grown faster, as I need to change the medium each two days. By the end of a week I have maximum confluence after split.
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