I’m working with fibroblast cells that we plate on a carbon nanotube surface for gene transfer. At the end of the experiment I’m trying to remove the cells with trypsin (or TryplE), but it appears that cells are getting ripped when washed off the surface, and I’m only recovering about 10% of the cells that are plated. I can visualize the cells on the surface prior to trypsinization, so I know the cells are present and viable.
Because of the uneven and fragile carbon surface I can’t scrape the cells. I’ve tried other varieties of releasing agent including PBS/EDTA, Detachin, and a few others I forget.
Any magic tricks folks have come up with to gently release cells that appear to have really made solid contact with a substrate?
How long are you leaving the cells in trypsin (or TryplE) and what concentration of trypsin are you using? You may need to leave the cells for some time in trypsin before they will detach from the nanotube surface. I have left cells in trypsin for 45 minutes to get them off a nanoparticle substrate. I monitor this under a inverted microscope over time. The cells were still viable even after that long incubation in trypsin.
That’s a very good point. So far I haven’t gone beyond 20 minutes with the TryplE. I could stain the cells with calcein-AM and monitor for release, and suspect that the TryplE was sufficiently gentle to go out to 60 min if necessary.
I could also try using trypsin in PBS/EDTA, although I assumed it might be a bit toxic for cells in extended incubations. What concentration of tryspin would you suggest?
I wouldn't recommend using the calcein-AM. Your cells will not grow after staining them with it. Are you able to see the cells attached to the nanotube surface and then detached/floating after TrypIE treatment? If you can gently tap the vessel holding your nanotubes that might help to dislodge the cells.
I find that using chelators doesn't work very well so I avoid it. If you want to try it, you may need to use 10mM EDTA for it to work effectively.
I always use the 0.05% trypsin. Less chance of damaging the cells in prolonged exposure. You could also try Accutase which is more gentle than trypsin and it does not need to be inactivated.
Fair point on the calcein-AM. I could try a cell-tracker dye or Hoechst stain. The difficulty is that our nanotube device is an opaque disc. I can image the cells from the top with a fluorescent Zeiss Axioscope microscope, but I can’t seen the cells without labeling them. I wouldn’t stain for cells I was going to work with downstream, but for working out the method I need to stain the cells.
I could try 0.05% trypsin, as well as TryplE and the accumulated releasing agents we have collected over the year (Detachin, Accutase, and a few others I forgot), just increase the incubation time as you suggested.
I think increasing the incubation time is the key. Not so much the reagent used to detach the cells. Stay away from the collagenases though, those enzymes will damage the cells. You may need to go longer than 45 minutes to get the cells off the substrate. In any case, after detachment, you can always take some and plate them in a small dish (24/48-well plate?), let them sit overnight and then see if they attach to the plastic to determine if they are viable/the incubation time was too long.
People think that cells are fragile and easily damaged (a few are indeed that way) when in reality they are quite tough and resilient.
Good luck!
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