When we FACS sort our CRISPR knock ins the low efficiency often means we get between 50-500 cells per sample. They go into a 96-well for recovery. They survive fine after sorting but they just grow so slow from lack of that cell-cell contact. Any tips on how to make them recover and grow faster? I remember an old flow core manager telling me people will often sort into 100% serum and grow them in serum only. Does that help? There's an insane part of me that thinks maybe I can take media from a confluent plate of un-edited cells, sterile filter it, and apply it to my sorted cells so they can get whatever signals that tell them they're not alone sitting in little island
Hi Julia, there's no easy shortcut around. I never tried growing them in 100% serum but I guess this wouldn't be alright for every cell type.
If this is really an issue you might consider generating a stable Puromycin resistant cell line prior your Cas9/sgRNA delivery. Then sort single cells on a feeder layer of the parental cell line treated with cytocalasin b (this would create a feeder layer of parental cells unable to divide) but providing that "contact" boost to your sorted single cells. After expansion (you'll probably need to provide feeders for 2 to 3 rounds of amplification) you can apply Puromycin selection, get rid of the feeders, and recover your enriched sorted cells. Honestly I don't know if it's worth the effort btw, and if your cells are just taking longer to grow, I would definitely go through the pain of waiting for them... Unless this is an experiment that you will repeat so often that this optimization would be worth.
Of course if you're using Lentivectors to deliver Cas9 and sgRNA, simply pick one with a Puro resistant marker and you could spare the effort of generating a resistant cell line beforehand.
In alternative filtering and adding the conditioned media from confluent cells it's not a crazy idea, and although that might not be enough since cell-cell contact is still not provided it's definitely worth a try.
Hope it helps
Francesco
I'm not sure what cell type you're using, but usually if I run into this problem with HPAECs (which really need their neighbors in order to grow), I make sure that I coat the plate I'm putting them in with whatever coating is appropriate for them (for HPAECs, it's gelatin/fibronectin; for HEK293s, it's poly-L lysine). This makes sure their surroundings are conducive to growth. You can also try, depending on cell type, to supplement your growth media with not only serum but glutamine/glutamax as well. These two protocol edits usually help grow my cells up enough so I can work with them in a few weeks' time. Worst case scenario, you can put them into an even smaller sized well and painstakingly transplant them as they become confluent into larger wells.
Hi Julia Ngo
I agree with the others in that you will need to ensure your cells attach (providing they are adherent cells) by coating the plate and that using conditioned media can be a good alternative.
I recently performed single cell isolation of HEK293T cells resuspended in conditioned media which I aliquoted into 96-well plates - the cells divided roughly every 24 hours and seemed to lose little growth potential.
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