I have cultured these cells before and they are very frustrating.  I did have much better success using ATCC's formulation.  The more sodium bicarb you have in your media, the more CO2 you need to have so that the pH remains neutral.  If you have 2.2 g/L of Sodium Bicarb, then 5% CO2 is what you use.  This is a very sensitive balance.  Also, sometimes the fetal bovine serum needs to be tested to see what lots of FBS work better for your cells.  Companies should be able to provide you with free samples to try.  Once the cells are stressed, then they are changed at the molecular level.  I suggest trying another frozen vial with the medium that ATCC suggests and bring them up slowly.  If you have any questions, please contact me directly at [email protected].  I hope this was helpful!!

Hi Lisa,

thank you very much for your reply, I really appreciated it. your suggestion is quite helpful, I will try to reduce the sodium bicarbonate concentration in my medium and see if there is any improvement. As you said, the molecular level is changed when the cells are stressed. so I am hoping to get another frozen vial from ATCC. But it 's quite EXPENSIVE, we've paid 500 pounds already on last vial, I am afraid we have to pay another 500 pounds on the new vial.

Hello Yuming,

I completely understand your frustration about stressed cells and the cost of replacing cells that are changed/dead/contaminated, etc.  Alas, this is the part of cell culture that makes things somewhat difficult!  I do have one other thing to offer, if you use the ATCC media and lower Sodium Bicarb, and your new cells are still not behaving, contact ATCC and tell them that you have followed all of their directions.  It could be that the lot they sent you contains cells that are not very viable.  This doesn't happen often, but it does occur.  I have pushed the issue a couple of times and always received a replacement vial of cells.  Just a thought!  :-)

Hi Lisa,

fantastic suggestion! I will definitely try to grow the new cells as the way in their protocol. it's not like we want to have replacement or complain to ATCC about issues, it's just waste of time, that really matters, especially when your postdoc contract is close to finish and you are keen to get publications ASAP :(

I know time is precious isn't it?  One more suggestion that may or may not work:  split your cells into a T-10 or 12-well plate and allow to grow.  It could be that the density in your flask is too low so you might want to reconcentrate.  Also, you might want to check for mycoplasm contamination.  Good luck!

Exactly! I've already done a passage from T75 flask to T25 flask in order to concentrate abit just like you suggested, hopefully they will grow more happily. Mycoplasm test, good suggestion, I will do it. Thank you for all your kind responses.

There are two types of cell populations in SHSY5Y.  Mixed adherent and cell suspension. When you subculture you should not discard the supernatant away. You spin it down  with the adherent layer that you trypsinize otherwise your adhrent cells will die. Best wishes

Thank you louiza. The weird thing is I have some late passage SHSY5Y cells which I have been growing for 2 years so far, I never keep the supernatant every time when I passaged the cells. I can still get RA induced differentiated cells from the late passage SHSY5Y cells. The reason I am currently trying new cells from ATCC is that I got feeling that my late passage cells have got S-type cells overgrown which affect differentiation efficiency.

Yes it is a good idea to try a new batch of cells. Phenotypic and global gene expression profiles  are likely to change in highly subcultured cells.

For the culture of SH-SY5Y DMEM:F12 from ATCC works best.

DMEM with 3.7% sodium bicarbonate is designed to culture cells under 10%CO2. You could either try to raise your CO2 to 10%, or lower you sodium bicarb. ATCC DMEM is the only one provided with 2.2 sodium bicarbonate, but quite expansive. I had experienced exact same problem with another cell line.

good luck

I use EMEM(Lonza):F12(Life Tech) supplemented with 1.2g NaHCO3, 10%FBS, 1% anti-biotic. Works like a charm. Also passage at 60-70%. Typically it will take 4-5 days to hit confluency from an ATCC vial. After that they will start to detach from the flasks.

Thank you, Martin. But if the cells start to detach from the flasks after 4-5 days in culture, how would you be able to differentiate them in dishes? I suppose full differentiation by RA will at least take 1 week to 10 days. perhaps once the cells start to differentiate, they won't detach from wells/dishes anymore.

SH-SY5Y will grow on top of each other if they get too confluent. So once you start seeing a good deal of non adherent cells just floating around in your culture, then you need to passage. If you don't, the cells will become stressed and they won't grow well.

Once they differentiate they are post-mitotic. So at that point you don't have to worry about passaging. Also you want to differentiate them at lower densities; otherwise, they wont form neurites.

The way I typical do it is:

1) Prepare a 6-well plate with basement membrane of your choice. I'd suggest 10ug/mm^2 of collagen.

2) Passage your cells when they are close to 60-70% confluency

3) Plate 300k cells for each coated 6 well prepared with 5mL of complete media.

4) Add your RA solution to the well (see below).

5) Let sit for two days, then replace media. By day 4-6 they will be largely differentiated.

To make RA stock, I store 100ul aliquots of 133mM RA dissolved in DMSO.

Right before I need it, I take the 100ul aliquot and dissolve in 50mL of DPBS. Be sure to protect from light and wrap in tinfoil. The working RA solution is now 266uM, so add 188uL of the 266uM solution to each 6 well.

Thank you very much, Martin, your suggestion is very helpful. just one more question, 300k seeding density in 6-well plate for differentiation sounds a bit high to me. I used to seed only 50-100k cells. When you replace the medium with differentiation medium containing RA, did you keep serum in your differentiation medium? and how much? thank you!

 I typically seed based upon these numbers http://tools.lifetechnologies.com/downloads/Useful_Numbers_Y14472_Useful_Nmbrs.pdf . it's for HeLa cells, but sh-sy5y are similarly sized. but you can certainly seed lower if all you want to do is differentiation. though i have read else where that high densities increase spontaneous differentiation, so a higher seeding density may aid this process a bit. not entirely sure. you should just test some serial dilutions and see what works best for you.

also, i keep serum constant at 10% FBS. i just add concentrated RA to the well and then dilute it by swirling around the plate.

Fantastic answer, thank you, Martin.

Hi Yuming, I have the same problem than you with my SH-SY5Y cells. Recently I have bought a vial from ATCC and cells don't expand and they are detaching from the flask. As you, I change the medium ever 4-5 days and flotating cells appears but adherent cells grows slowly. I use DMEM/F12 GLUTAMAX (GIBCO), 10%FBS, P/S. Are your cells now grow ok ? Have you any suggestion for me? 

I have the same problem with the ATCC stock. The cells detach after 3 days of adherent growth. I have found similar behavior in both DMEM only and DMEM:F12. Help!!!!

I also found the same problem Subramaniyam. In my opinion 2 days of culturing is the best for me. The floating cell is not much and also the adherent cells still can grow when you do subculture.

Hi Novi,

I experienced the same problem even after 2 days of culturing. When I tried to subculture the cells they would not grow and started to float. Is it possible for you to share some micrographs of your cells?