SH-SY5Y single clonal expansion

[Jerry Xu]

I'm trying single clonal expansion of SH-SY5Y after showing CRISPR works in bulk DNA.

Basically by serial dilution I first diluted into 1cell/well of 96-plate. Wait for a week, then I could see "cell clusters" in 20% of all wells. 

Then I digested those cells (to make it spread-out) and transfer to another new plate.

But lots of cells die or don't grow well.

I'm wondering if anyone here is experienced with SH-SY5Y single cell expansion? Maybe this type of cell is hard to grow from single cell? Any tricks?

Thanks

[timjsargeant]

Hi Jerry

We tried for all of 2015 to make monoclonals out of SH-SY5Y cells for CRISPR KO. We were able to generate monoclonal knockouts for one gene, although as you say it is very difficult to keep them alive. We tried a lot of different ways but got best results when we 

1) transfected GFP CRIPSR construct in with lipofectamine 3000 (about 2% of cells were positive)

2) sorted for positive cells (typically got 2000 cells per transfection)

3) plated these cells very sparsely in large tissue culture dishes (from memory, a couple of hundred cells in a 10 cm dish)

4) waited for colonies to form and then used silicone grease and cloning rings to isolate monoclonal populations

To increase survival of cells we had to use DMEM:F12 media 1:1 with 20% fetal calf serum and we also added some filtered conditioned or 'used' media from a stock SH-SY5Y flask. We added about 1 ml of this stuff to the media of a 10 cm dish. We heard another lab had increased survival by adding a bit of filtered conditioned media. I guess it makes the cells think they have a lot of friends in the same dish. We never tested this properly with a control but our success rates went up after adding conditioned media, with 20% FCS.

Good luck! I work with HeLas now!

Kind regards

Tim

[Jerry Xu]

Sorry for late. I've been away for other research and just back for CRISPR again.

What a nice work and analysis!

But still, I'm quite confused, what for the first week SH-SY5Y grow quite well from a single cell (obviously this suggests they don't need "friends" or aggregates from beginning to grow); but once I did digestion using trypsin they simply die. Is it because I digested too long, ie. should I digest cells very quickly since they are very few in number and fragile to trypsin?  For each 96-well I used 10-20 ul I think.

[Tim Vierbuchen]

Hey,

maybe the usage of trypsin is too harsh for the cells and/or the digestion time is too long.
You can try to use other reagents that claim to be more "specific" for extracellular matrix molecules or whatever (e.g. Accutase). Pre-warm everything to 37 °C, wash the cells with PBS before adding Accutase and try to keep the time frame for detachment as low as possible (--> check with microscope).

Best
Tim

[Jerry Xu]

I think so too.

1. Why pre-warm everything, you mean including trypsin/Accutase? I never pre-warm such enzymes just in fear their enzyme activity will go inactive.

2. ''Keep" the time frame for detachment as low as possible", you mean very quickly add culture medium after digestion?

[Tim Vierbuchen]

I never worked with this cell line but as you describe it, these cells seem to be very sensitive.

1. for example if you remove the medium, add cold PBS, then cold Trypsin and put the cells back to 37 °C, the temperature drops very fast for the cells and quickly goes back to 37 °C. As I said, I did not work with these cells but maybe they don't like these quick temperature shifts and it's too stressfull for them especially when the cell number is quite low. I mean you can just try it.

2. Yes, don't leave the cells too long with the trypsin/accutase. As soon as you see that most of the cells are detached you should add the culture medium.

[Carlos H. V. e Vieira]

Hello everyone,

I´m also interested in making CRISPR KOs in SY5Y cells but I got no experience with that. It was mentioned that adding conditioned media helps to keep clones alive. Could you provide us with more information on how you obtained and used CM? Is it just the media from exponetial growth SY5Y cells filtered and stocked at -80°C until use? What proportion of fresh/contioned media would you recommend?

Also, I´m using RPMI media with my SY5Y cells. Do you think it makes a difference? RPMI 1640 contains biotin, vitamin B12, and PABA which are not found in DMEM, so it´s nutrient richer...

https://www.thermofisher.com/order/catalog/product/42401018

[Fulvio Celsi]

Hi Jerry...

just saw your question...so, we are doing serial dilution in SH-SY5Y and it is tricky...basically, if you go to a confluence lower than 40-30% they are not happy at all and they die soon. So what we do is to wait until they reach 90-100% confluence in a single 96-well (basically, 3-4 weeks from  a single cell) then move it to a 24-well, then 12-well..and so on...until they are expanded. It is slow but it works...also we grow it in DMEM:F12 10%FBS 1%Non-Essential Amino Acids (NEAA)...

good luck! 

[Jerry Xu]

Thanks Fulvio

I'm back to CRISPR again.

You mentioned "to wait until they reach 90-100% confluence in a single 96-well"

My observation is the single cell will form small clump, stacking over each other, and will die if you don't digest them with trypsin.

So you may use any trick to make them grow into the whole 96-well?

Thx

[Jerry Xu]

Hi Tim

I'm wondering what plasmid you use for SH-SY5Y CRISPR? Is it PX458?

Thx

On Wednesday, February 17, 2016 at 5:29:04 PM UTC-5, timjsargeant wrote:

[rie...@gmx.net]

I am joining this discussion late but still hope to add in some recent wisdom. I am currently trying to generate single CRISPR HDR clones. While this is still in progress I did some preliminary experiments using untransfected SH-SY5Y. I tested several medium conditions and this is where I am currently at:

I select single clones using flow cytometry. So basically FACS but sorting cells not by fluorescence but only by forward and side scatter to identify individual cells. Our core facility sorts individual cells directly into 96well plates (containing 200 µl medium) which just works perfectly. For post FACS medium I tested different serum conditions (from 10-35% FBS) in DMEM High Glucose supplemented with or without conditioned medium. A few of the results are listed below: 

(1) 150 µl 35% FBS + 50 µl conditioned 15% FBS => more than 50% survival rate 

(2) 200 µl 30% FBS => about 30% survival rate

(3) 200 µl 25% FBS => about 20% survival rate

(4) 200 µl conditioned 15% FBS => no survival

I will now proceed to the full experiment, i.e. CRISP transfection > enrichment by Puromycin selection > single cell sorting. For the full experiment I plan to use 150 µl 30% FBS + 50 µl conditioned 30% FBS. If any one is interested I will post the survival rates once I have them. May take a few weeks from now though.

[Aaron]

Hi Jerry,
Have you consider using a lentivirus for Cas9 expression. The lentiCRISPRv2 works well !!

best,
Aaron

[Peter Zeng]

Hi friends, 

Would you mind telling me how your CRISPR transfection in SH-SY5Y worked and your puromycin selection condition?

Many thanks,

Peter

On Tuesday, July 18, 2017 at 3:07:08 AM UTC-4, rie...@gmx.net wrote:

[Rie...@gmx.net]

I will gladly do, but it is still ongoing.

Currently second day of Puromycin selection (0.5 µg/ml). Single cell cloning due next Tuesday.

I will get back to you in 2-4 weeks I guess.

[Kyra]

Hello! I'm also doing single-cell expansion of transfected SH-SY5Y and am curious as to whether the protocol with 150ul 30% FBS and 50ul 30% FBS conditioned media worked for your transfected cells?  

[Rie...@gmx.net]

Sorry, I promised an update and I will give a proper one in due time. 30% FBS works perfectly, I now use it routinely. Though for simplicity sake and a little added boost I switched to just 200 µl conditioned 30% FBS DMEM High Glucose. Medium is conditioned over night on a 50-80% confluent plate. Clonal survival is around 50-60% which works for me. If you sort to 96 well plate, you will likely see no cells for 2-3 weeks. After 3-4 weeks you will be at 50-90% confluence of the 96 well.

I transfect on a Wednesday, on Thursday I add 1 µg/ml Puromycin and 7.5 µM RS-1 to increase HDR. Friday I switch back to normal medium (I use DMEM High Glucose + 15% FBS) and on Monday I do single cel flow sorting into 96 well plate based on forward/sideward scatter. I directly sort into the conditioned medium. The reason I haven't posted an update yet was my struggle with ssODN-based HDR and RFLP-based genotyping. I now abandoned that strategy back to classical HDR targeting vectors with 0.8-1 kb homology arms and just this week got my first hetero- and homozygous knockin clones. So it definitely works.

 

 

[Mark]

Hey guys, any advice on isolating single SY5Y cells? Trypsin digestion gives me a lot clumps that can't be dissociated even after trituration. What enzyme/procedure would you recommend?

Thank you !

[Xu Zhang]

Hi! I used trypsin digestion which also gave me some clumps. Then the FACS staff filtered the cells to remove the clumps, and it worked fine for me. If you have a good transfection rate and can afford to lose some cells, this should work. I am now struggling with the single cell expansion. Thank you for all the good tips here, I will try the 30% FCS and condition medium protocol. 

在 2018年3月14日星期三 UTC-7下午1:17:40，Mark写道：

[Lydia Bartsch]

Hi!

I am trying to do CRISPR HDR experiments with ssODN in SH-SY5Y using RNP complexes, so I do not select with Pyromycin but check my clones with sequencing. Does anyone know if I can use Pen/Strep in my medium for single cell clones? I observed that wildtype cells grow much slower with Pen/Strep. Do cells with an introduced LOF mutation grow at all?

Also, what size of ssODN did you use? I have 90 bases and I feel like HDR is not very efficient in my cells.. What are your experiences?

Thanks in advance!

[Mona Khazaei]

Dear  Rie,

I have one question regarding conditioned media. After cell sorting in 96 well plate how often did  you change media? and did you always add again conditioned media after removing old media or you switch  back to normal media?

Regards,

Mona