Hi Benjamin,

I cultured SHSY5Y cells without any coating at all, in fact, culturing Poly-L-lysine coating resulted in massive cell loss... I differentiated the cells for 5 days in 25cm2 flasks with retinoic acid added at seeding with 50/50 Medium (medium: 50/50 F12 Kaighns/DMEM + 15% FCS) replacement after 2-3 days. After 5 days I trypsinised the cells and seeded them with a new dose of RA in smaller dishes for microscopy. This worked well.

Hi Benjamin,

I would suggest, that you do not exchange media, but slowly add extra media with 2x concentrated TPA e.g. at a 1:1 ratio (100µl media : 100µl media+2xTPA) so you will end up with the final concentration wanted. This could avoid shearing forces on your cells and hopefully will not detach them from the substrate. Spining the 96well plate at 150g for 10min prior to aspiration of media could also help keeping the cells attached. This might avoid the use of matrigel or poly-L-lysine.

Best Kai

Hi Benjamin,

Could you differentiate them in larger sized culture plates, and subsequently plate differentiated cells in 96-wells for your assay?

I've differentiated them in 6-well & 12-well plates, with excellent results. And this was without additional coating..

Reeteka

Thanks for your answers guys.

@Kai - Have you done this with these cells? I have considered this, but since it is a 7 day differentiation protocol, I am concerned about leaving the same media on there that long.

@Reeteka - I don't really know if it would affect the assays. After having differentiated the cells in the larger plates, have you ever tried re-plating them?

Hi Benjamin,

7 days might really be to long, because you probably don't want to measure nutrient starvation, right?

So another option could be to only partially exchange media, or using 200µl in the begining and add of 50µl 5x conc. TPA for the stimulation. Did you check if you might have to many cells at the start? Perhaps a reduction of starting cell number/well helps.

Best Kai

Hi Benjamin,

Are you using 96 wp because you need a high throughput assay system? I have tried differentiating SH cells on 24 and 48 wp and it worked well, without lysine or other coating. Also added removed 50% conditioned and added 50% fresh media rather than a complete media change.

Agree with @Kai, how many cells are you plating?

Good luck,

Claire

Hi Benjamin,

I have also tried SHSY5Y cells to grow in 12 well as well as 6 well plate (uncoated) and also I have seeded them again after trypsinization..and they were fine...media was EMEM:F12(1:1) with 10% FBS or you can also use DMEM(high glucose) with 10% FBS.

But addition of some more FBS (around 20%) at the time of seeding will help the cells to attach firmly.

Hi Benjamin,

I cultured SHSY5Y cells without any coating at all, in fact, culturing Poly-L-lysine coating resulted in massive cell loss... I differentiated the cells for 5 days in 25cm2 flasks with retinoic acid added at seeding with 50/50 Medium (medium: 50/50 F12 Kaighns/DMEM + 15% FCS) replacement after 2-3 days. After 5 days I trypsinised the cells and seeded them with a new dose of RA in smaller dishes for microscopy. This worked well.

Hi Benjamin,

I also differentiated the cells using retinoic acid or retinoic acid plus BDNF and it worked well.

Hi Benjamin,

Cells can be re-plated and that works fine. Though remember that once they start differentiating, their life-span is limited.

Reeteka

Wow, thanks for all the responses. This was my first question posted to RG, and this is great!

I guess I should provide a little more info on what I'm doing:

I am a medicinal chemistry student looking for a relatively high-throughput model system for studying the effect of compounds on dopaminergic neuronal function. SH-SY5Y seems to be the way to go, and although the literature is a little spotty, there's seems to be some agreement that RA treatment with FBS decrease alone causes differentiation toward a cholinergic phenotype, while a subsequent treatment with either BDNF or a phorbol ester directs the cells towards a dopaminergic phenotype (ref: Hong-rong et al. Chin Med J 2010; 123(8):1086-1092). I'm currently working off the most cited protocol for this procedure which calls for Day 1 aspirate media and replace with 5%FBS+10 uM RA, Day 4 aspirate media replace with 5%FBS + 80 nM TPA, allow 3 days then do assay.

I'm plating 10K cells/well in 96 well format and 60K cells/well in 24 well format. For the wells that stay attached, this seems to give me ~80-90% confluency by then end of the differentiation process. For the wells that stay attached anyway.

If anybody has anymore advice....please do share. In the meantime, I'm going to go get started on trying your recommendations and combinations thereof. THANKS!

forget to mention before, ...be careful about the passage no..usually I use within 20 passage after I got the cell from ATCC.

Hi again, 10K per well seems a little high, especially if you are plating them in non-diff media to settle them before differentiating. Do you think they look overcrowded? It might be a reason why they are lifting.

Hi Benjamin,

There's plenty of good advice above but just thought I'd add my experiences with SH-SY5Y for what it's worth. I've had real trouble with them in 96-well plates (sometimes with significant cell loss even after fixing) and they seem to behave much better in larger well sizes. So, the advice about half media changes with reagents made up at 2× final concentration seems a great idea. Claire's comment is correct too, as they will start to detach one they get confluent. You could experiment with lower seeding densities to compare.

Finally, if you're looking for a dopaminergic model, the rat line PC-12 has quite frequently been used as well. We've found these grow fine in regular 6-well plates, although I can't comment on smaller well sizes in this case.

Good luck, hope you get everything working OK!

Mark

Hi Benjamin,

I´ve used SH-SY5Y cells for quite some time now, and I tried different protocols to differentiate them into a dopaminergic postmitotic status.

First of all, have you checked them for micoplasma?? I had the same issues you describe for a few weeks, plus the cells would not resist transfection, and when we checked we had micoplasma contamination. Once we started working with a new set of cells everything was ok.

Regarding coating, I never coated the plates and used normal 6 well, 24 well and 96 well plates.

Then, from the 3 differentiation protocols I assessed (RA alone, RA then BDNF, and RA then TPA), the RA/TPA protocol was the most effective as assessed by WB of dopaminergic markers (TH and DAT). I kept 10 % FBS, because when I tried to reduce it cells didn´t look very happy.

- The differentiation protocol I used consisted of: plating the cells at quite same large dilution (1:8 or 1:10) in RA 10 uM. Leave them for 3 days, then lift them and seed them in medium with TPA 80nM at 12000 - 13000 cells per mL. I would use 200 uL for 96 well plates. Leave them for 4 days in TPA, then do my assays or transfect them...

I hope this is helpful and good luck!

elena

Hi Benjamin,

There is lots of good advice above - my own experience during my PhD if it helps:

I agree with the comments about confluence, you need quite low cell density (40-50%) to achieve proper differentiation, to encourage the cells to reach out to one another and form contacts.

After FBS was removed the time before they started to float off was 2-3 days in my experiments in 6- and 24- well plates - more dense cells stay adhered longer. It's a balance between enough cells and not too dense! I used dibutyryl cAMP without FBS (following papers by Flaskos et al) to differentiate both N2a and SH-SY5Y with success.

If you have to change the medium, aspirate gently from the side of the well.

My suggestion would be to try a few different densities that reach 50% confluence in normal growth media in about 2-3 days- actual numbers per well depend on how you count the cells- then remove FBS to start differentiation & try different RA or dbcAMP concs (1-5mM?) and time periods between 1-3 days to optimise the number of healthy cells.

After differentiating I never tried re-plating (might cause cells to change morphology again?) but this might be possible if you could measure a differentiation marker before your assay - e.g. dopamine???? or gene markers by rt-PCR to ensure differentiated characteristics are retained.

Good luckl!

Abi

P.S. Re - Kalpita's comment - I agree, keep the passage number as low as possible, below passage 20 is a good guide.

Hi Benjamin,

SHSY5Y cells have been known to change their profiles after a large number of passages. So as Kalpana mentioned, never go above 20 passages.

My experience with SHSY5Y cells - They adhere to the plates without any coating. However, when needed for imaging on glass coverslips or so, we use fibronectin to coat glass dishes. 10 ug/mL fibronectin for 6-12 hrs works pretty well. You can try that in your system.

They seem to be very sensitive. So media changes are very gentle and immediate.

It is great to have such a discussion, especially about the cell lines and their behavior and people's suggestion. I think we should have a section of cell line behavior with things to keep in mind. :)

@ Abigail Dalzell: I have been wondering why is it necessary to remove FBS/serum during differentiation?

Does anyone have experience with this cells and Hypoxia? How many hours are necessary to have more a less 50% of death?

Thanks

Brigette, broadly serum has a variety of growth factors in it which help drive mitosis. By removing these factors, you're changing it's expression profile, and driving it towards a differentiated state.

Claudio, I'm not sure on the specifics, but these cells are quiet commonly used for hypoxia studies. I'd suggest just doing a very general scholar search for hypoxia in shsy5y.

Hi, I also have a similar problem with these cells. I usually differentiate them in 6-well plates coated with collagen. (As advised by Martin). I use the vacuum pump to draw the media and 1ml pipet to add fresh media. Unfortunately, with every media change I lose cells starting from the edge. I tilt the plates and go very slow. Still by 2nd or 3rd media change, half of the well is clear of cells. I think cells are detaching while I am aspirating old media, as well as when I am adding fresh media. Is it a common issue with these cells? Please advise.

Unfortunately pretty much all cells like to detach. Maybe either turn down the vacuum pressure or use an air displacement pipette where you can control the speed. Be careful not to change the media too often (more than necessary). Also if they have no FBS in the medium they will detach much quicker. If you are doing a long incubation to differentiate the cells (more than 2 days) I would consider putting FBS back in at a low % e.g. 2.5%, even 1%. Obviously you would have to validate this to make sure you still get the full differentiation effect. Poly-L-lysine is the coverslip coating that I used with success when differentiating these cells with dibutyryl cAMP, but I was only incubating for 24 hours and I don't know how comparable this is to collagen.    The first thing I would try is to take away the vacuum pump; also check you've got the right confluence (c.60%) for differentiation.

Good luck!

Thanks Abigail..

Yeah, I have been thinking of using a vacuum valve or something to control the pressure. I am not aware of the sir dispenser pipet, but I will look those up too. I just dont think it is feasible time-wise to use pipets to aspirate the media slowly. Vacuum is much faster. If I pipet, I can go very slow, but then by the time I will be done with all the wells, they will start drying up, which isn't good either. 

Thanks for the info. I will look into it. 

Are you aware of any vacuum control valves?

If the cells are not dying until addition of TPA..your problem may be the TPA solvent. My guess is you are using DMSO. A max of 0.6% DMSO final concentration is the most these cells can tolerate, before reaching toxicity. But I usually keep it below 0.5%. This means you can dissolve the TPA in DMSO, and the 2nd and 3rd dilutions in media. You will have to optimize this part. Also, include a DMSO control column in your plate.

HI, Shruti,

It's less about going slow, in my opinion, than about force applied. You could go reasonably quickly with a 5ml tip in air displacement pipette as the tip aperture is quite wide. Take care to run the fresh media down the side of the wells as you apply it as this takes some of the pressure off too, and still tilt the plate. As far as solvent goes, keep the DMSO below 0.2% if poss (as Birgitte says, this needs optimising). Also, consider how long you are incubating them for vs how often you change the media/ and/or adding in a very small % of FBS to support growth if it's for a long time once differentiation is underway. My experience of vacuums is limited and I don't know of any valves, but can you just turn the pressure down? Yes, definitely include DMSO control otherwise you have no true control for any toxic effect.     

Good luck!

Hi, Benjamin

Did you already solve your problem? I also have the same thing now when I am working with differiented shsy5y cells. Using 96 well plate with 10k cell/ well after 1 day plating cell with grow medium in 10% FBS (the density of cell after 1 day arround 50% only), I replace all medium with 1%FBS and 10uM RA but some cell detach during changing medium. Could anyone give me some advice about that because its look well in 6well plate but I need 96well for my furture assays. Thank you so much

Hi Dang,

You can use a Corning CellBind plate. Flicking the media out instead of aspirating it helps a lot, too, but you have to be extra careful about sterility.

Best,

Ben

Thank you so much Benjamin.