Dear All,
I am taking over the Master's student's project and I am a new PhD student. We have experiments involving tumour implantation in mice. The MSc student is used to scraping cells down when passaging and only uses trypsin to detach the tumour cells for implantation.
In my previous lab, I was taught to trypsinise as it is less damaging to cells when passaging and that apoptotic cells could influence culture environment. Therefore, I have been using trypsin (5 minutes, 37 degree C). However, I have noted a difference in cell growth as the MSc student is used to passaging at very, very low concentrations of 1:1000 whilst I resorted to passaging 1:10 every 3-4 days. I have been keeping track of the growth rate of my cells which is about 3.1-3.7% (quite consistent) but they seem to be growing much slower than the MSc student.
I consulted the MSc student and I was told to swap to scraping. I am just curious as to how this may affect the cells and whether anyone has ever noted such a huge difference.
Note: We re-use the T75 flask (hence, why I decided on trypsinising to cause less damage to my cells).
We mostly run FACs and IHCs in our lab if that will help determine which method would be more suited to our cause.
Thanks.
Hi, Darellynn Oo.
I do agree with Arseniy Yuzhalin. Do use accutase instead of Trypsin and see If it improves your results.
About the scrapping, my feeling is, some people do it better than others (no offense) and then cells are only membrane damaged and the machinery is able to fix it. As tumor cells are metabolically highly active, it works fine.
Enzymatic treatment may go a little further in damage. But this is only an opinion.
If you check papers about this subject, you will find some support for trypsin instead of rubber scrapping like in this paper Anticancer Res. 2018 Dec;38(12):6669-6672. doi: 10.21873/anticanres.13034.
But ATCC reminds us of some situations where cell scrapping is better.
http://www.atcc.org/Global/FAQs/5/2/Scraping%20cell%20cultures%20instead%20of%20using%20trypsin-569.aspx
So, try accutase or rubber scrapping and check it.
Hope it helps a little.
All the best.
P.S. Check this other discussion associated with the same kind of problem ... https://www.researchgate.net/post/What_is_the_difference_between_scrapping_and_trypsinising_cells_for_extracting_protein
In some cell lines more time required for trypsinization, scrap gently just to remove not to rupture cells. No problem.
Hi Darelynn,
In my experience if you leave EO771 with trypsine for too long, they lose their adhesion proteins and they are not able to recover them. You will notice that cells are alive but they never atach to the plate. For this cells, 5 minutes is too long. I leave them in trypsine for 1 min at 37 degrees to do the passages and it works just fine.
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