I do cultivation of human mesenchymal stem cells on different (hard) porous scaffolds. Since I want to quantify DNA and protein content I have to detach and disrupt the cells (for now it would be denaturated protein is ok). Standard procedures that work on cell culture dishes like the detachment with accutase or trypsin doesn't work, cells keep attached. Any ideas about that?
In parallel I want to lyse the cells directly on the scaffold (without prior detachment). We already tried different lysis buffers with collagenase I and II, hyaluronidase as well as combinations of them. What I want to try next are different freeze-thaw cycles, sonication in water bath and sonication with a probe.
Is there anyone having the same problem and already established a solution or has other approaches for that?
Thanks a lot.
To detach cells from the scaffold, you might try 30min with trypsin. You remove the trypsin and add a solution of collagenase I for 1 to 3 hours. The first enzyme is used to increase the access of collagenase to collagen.
You could try a mix of collagenase and dispase (ROCHE sell a mix of both). The time of incubation is 30min to 3hours. It depends of the extracellular matrix.
I do not know the composition of your scaffold but you could try to freeze it in liquid nitrogen and cruch it in a mortar. The mortar must be kept in liquid nitrogen. The powder must be recover in the lysis buffer and centrifugate in order to separate the powder of scaffold and cell lysate.
To detach cells from the scaffold, you might try 30min with trypsin. You remove the trypsin and add a solution of collagenase I for 1 to 3 hours. The first enzyme is used to increase the access of collagenase to collagen.
You could try a mix of collagenase and dispase (ROCHE sell a mix of both). The time of incubation is 30min to 3hours. It depends of the extracellular matrix.
I do not know the composition of your scaffold but you could try to freeze it in liquid nitrogen and cruch it in a mortar. The mortar must be kept in liquid nitrogen. The powder must be recover in the lysis buffer and centrifugate in order to separate the powder of scaffold and cell lysate.
To remove cells from 3D culture I usually use trypsin for 20 minutes and I am getting good results too. I think you should optimze trypsin exposure time.
Thanks a lot. I already tried to use liquid nitrogen and a mortar but it didnt work ( very poor yield). I'll try to increase incubation in trypsin/collagen.
Does anyone have experience using freeze-thaw cycles or sonication for this purpose?
I did the same in my Diploma Thesis. We were successful in lysing the cells with a supersonic probe. If you wish I can send you the Methods-Part.
We have used gentle sonication for 5-15 minutes on our nanofiber scaffolds with good success. You can use sonication in combination with any of the tyrpsin, accutase, etc. methods as well. To digest the cells while still attached to the nanofibers, we use DNeasy or trizol and they both work very well.