Hi everyone. I am trying to make point mutations in iPSCs using CRISPR/Cas9. I'm using lipofectamine stem transfection, and then waiting 48 hours before FACS sorting iPSCs (our Cas9 has a GFP) to then be isolated as single colonies.
After plating the cells after FACS sorting, I have extremely low numbers of cells attaching, like about 50-100 out of 10,000 plated into a 10 cm dish. I have tried optimizing the conditions and would like suggestions for things I could do differently.
I pre-treated the iPSCs before FACS for 2 hours with 10 uM ROCK inhibitor. i used Accutase to dissociate and filtered the cells into 5 mL FACS tubes. I kept the cells on ice while waiting to be sorted. I sorted the cells into 4 96-well plates and into a 10 cm dish (coated with hESC matrigel) to see which way would work better. The cells sorted into the 10 cm dish attached as single cells, but in groups close to each other which makes me think the colonies will merge before I can pick them. I used MTESR plus + 10% CloneR + Penn/Strep as the sorting media and followed the CloneR protocol for feeding - put them in a 37C/5%CO2/4%O2 incubator for 48 hours after sorting, then did a full media change with CloneR. I was expecting to see better survival with the CloneR but am not, in the 10 cm dish or almost any in the 96-well. Please let me know if anyone has suggestions!
Hello,
If you resuspend your cells in DAPI/PBS, I recommend resuspending them in their culture medium containing the DAPI and sorting them at 37 degrees. Also, while waiting do not keep the cells on ice. The coating might be a good choice but the risk of differentiation should be considered. Also, you would try the initial seeding with a low volume of media and after 3-4 hours you can add the fresh medium. Hope this helps!
Dear Stacey,
You mention you treated with 10 uM ROCK inhibitor 2 hrs before FACS. We do not use CloneR (ROCKi) or matrigel (instead geltrex). If the problem is only single cell survival then I would suggest maintaining the IPSCs in 10 uM ROCKi until a colony forms. I would agree with Didem Tecimel on not keeping your cells on ice.
Hi, did you consider to use a plasmid containing antibiotic marker selection, instead of GFP, for example puromycin? In that way you could skip the whole FACS sorting procedure, which is very stresfull for all types of cells. After the selection (24h incubation with puro) change the media and allow the cells to recover (it can take a few days) and then replate the cells using the reagent that will allow you to obtain single cells in suspension.
Hi,
We've recently launched an improved product to better support single-cell sorted cells, CloneR2. The protocol used is similar to CloneR, but we found that some changes to the formulation resulted in much better survival for the cells. While we haven't directly tested chemical based transfections, we would recommend the addition of CloneR2 to support post-electroporation recovery and then in the seeding media of your 96-well and 10 cm plate. Your particular cell line may be more sensitive to sorting, so we would recommend minimizing the Accutase treatment time required for your cells to form a single-cell suspension as well as keeping the flow rate on the lower end when processing your cells.
1) You could also try a higher plating density to improve survival after the GFP sorting. The cloning step with low density seeding or single cell sorting in CloneR2 can still be done at a later time, independent of sorting for GFP. This may improve survival after stressful transfection and GFP sorting, and the additional time can also be used to analyze gene editing efficiency and get an idea of how well the gRNA worked, e.g. by isolating gDNA from the pooled population once the cells recovered well, doing a PCR on the locus of interest and sending the product for Sanger sequencing for TIDE. (As the GFP expression does not necessarily correlate with gene editing efficiency.)
2) 48 h after transfection may also be a bit late to do GFP sorting, we see that much of our GFP-Cas9 has already degraded by that time. Your protein may be more stable, but it could be good to check on the GFP+ status of the bulk culture, and if 24h may be a better time point for the GFP sorting.
If you'd like more help or want to discuss further, please reach out to our Product and Scientific Support team at [email protected] and we'd be happy to continue to work with you to get your experiments back on track.
Good luck on your experiments.
Hi Stacey,
There are already some good ideas to try in the comments here. One thing I might recommend trying is a different plate coating material. GelMA is a great biocompatible hydrogel that can be mixed with a photoinitiator and curing to various degrees using different wavelengths of light. Its pretty good at providing better cell attachment than just plastic or matrigel.
OkaSciences makes a really good quality GelMA that is already premixed with the photoinitiator (either LAP or Igracure 2959) and ready to use. Plus they ship really quickly so there is very little lead time: https://okasciences.com/shop/okagel-liquid-2/
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