I need to remove the cells seeded on the scaffolds (PCL) without damaging them or compromising their ability to be tested using SGAG, DNA, and Collagen assays. We have tried just using trypsin (though for only about 5-6 minutes) and I do not think (though I'm not sure) that the scaffold pores are small enough to constitute a 3D environment for the cells.
Hi Karl,
Papain is milder than trypsin, you can also consider Collagenase or Proteinase K.
once we were run out of trypsin in the lab and I scraped/knock them off the flask.
Regards,
Amin
Hi Karl,
You might try papain digestion over night, or do 10-15 min digestion.
You could also try snap freezing the scaffolds and then go for mechanical distruption followed by digestion.
Anyway, if you doubt to have cells on it just go for a DAPI staining before and you will see if that is the case.
Cheers
Michele
Hi Karl,
Go for ultrasonication. you can get cell lysate for biochemical assay and your scaffolds will remain intact.
Cheers
KG
Thanks for all the feedback, but we have concerns about ultrasonication. Is there any significant risk to DNA damage using this method? We need to run DNA content assays as well as do some sequencing down the road and I want to be sure that this is safe.
Well Karl,
I did not checked DNA integrity after Ultrasonication. you can do a test run to check the DNA damage before going to ultrasonication with your precious samples.
regards
Hi Karl,
during my PhD I had to perform DNA, GAG and ALP assay on hMSCs seeded on PCL scaffolds.
My procedure was 3 times freeze drying and sonication afterwards. Some people in my group went for papain digestion along with a lysing buffer, I didn't use that method since I needed to check ALP activity and the papain would have it impossible to be performed.
Cheers,
Andrea
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