We have a quick (in vivo) biopsy to lab transfer time, we wash three times, use multiple types of medium with different concentrations of antibiotic, EGF, cholera toxin etc. Others in the lab have also used irradiated 3T3 feeder layers.
Any tips or is it just a matter of needing large numbers? We must have explanted 50 different patient samples by now.
Andrew, I've done this with oral SCC quite successfully (I don't know about their HPV status but I don't think that would matter). I think we collected about 45 biopsy samples from SCCs of tongue, floor of mouth etc. We managed to culture most of them and only lost a few through fungal contamination and a few that just refused to grow. (When I say "we" a lot of this work was done by Simon Broad, the lab manager and tissue culture guru from the Watt Lab, where I wrote my PhD).
I collected the samples from theatre and kept the processing simple.
1. Quick wash in PBS + betadine 10%
2. Quick wash in 70% ethanol.
3. Repeat wash in ethanol.
4. Rinse in PBS.
5. Digest in trypsin EDTA (0.05%) (you can leave it overnight at 4C or a couple of hours at 37C)
6. Chop it up manually with a scalpel.
7. Neutralise the trypsin with your culture media (We used FAD + FCS + HICE + P&S).
8. Spin 3min 1200 rpm.
9. Aspirate then resuspend in media.
10. Plate on irradiated J2s.
11. Change the media every other day. After 10-12 days you'll start to see colonies.
12. Once established most SCCs don't require the feeder layer.
We tried straining the digested tissue to get rid of the fibrous stringy bits, but essentially that's not necessary and might actually help to just leave some ECM in the mix!
Let me know how you go.
Hi Stephen,
Many thanks for your help - really useful. Will respond via DM. All the very best.
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