Hi Nese,

I work with BM-MSCs too and I have not had any problems detaching the cells with trypsin. Have you tried using fresh trypsin-EDTA (unopened bottle)?

After you add the trypsin to the cells you can put them in the 37C incubator and that might help them detach better.

You can try using Accutase to detach the cells. It is more gentle than trypsin and I always use it to harvest cells for FACS.

You could also try using cell stripper made by corning (catalog number-25-056-cl). I use it for BM derived cells. I think it is acidic in pH though, so be careful if your cells contain a fluorophore that you need for FACS.

sounds like you should try a fresh bottle of Trypsin-EDTA.  But, if your MSC have differentiated to osteoblasts and have started to deposit calcium, they will be harder to remove from the plate (likely only to happen at high density though)

We also detach BM-MSC easily, but after some cultivation time I usually have to use Tryple SELECT 10x - it works quicker, and you don't have to leave your cells in trypsin so long. EDTA ist very important, a fresh bottle is too - and you really have to watch they don't come too close to confluence.

I however do not have any experience what Tryple Select 10x does in terms of surface markers, sorry.

There are many protocols/papers out there that you can try specific to murine BM-MSCs:

1. http://www.nature.com/nprot/journal/v7/n12/full/nprot.2012.125.html

2. http://www.nature.com/nprot/journal/v4/n1/full/nprot.2008.221.html

3.https://www.researchgate.net/publication/51692232_Impact_of_bladder-derived_acellular_matrix_growth_factors_and_extracellular_matrix_constituents_on_the_survival_and_multipotency_of_marrow-derived_mesenchymal_stem_cells._J_Biomed_Mater_Res_A_100A72-83

As others have pointed out, the simplest explanation is your trypsin is old/bad.  Is it stored at the proper temperature (-20degC)?  TrypLE is fine at RT, so to still have a problem is odd.  TrypLE typically works in the same duration or 30-60sec longer than 0.25% Trypsin, still you shouldn't have to wait more than 4-7min ever.  You may want to double-check your volume to surface area ratio.  For a typical 10cm dish, I would recommend aspirating all culture medium, washing with 5ml PBS, one wash of 1ml trypsin, then 3-5ml of trypsin at RT or 37degC for 4-7min.  The third issue could be your confluence.  If they are overconfluent you may have more ECM to deal with and differentiation.  Typically below 70% confluence is acceptable. 

IIf you still have problems, you may want to post a picture of your culture, 10X and 20X would give a great idea of what you have. Goodluck.

Article Impact of bladder-derived acellular matrix, growth factors, ...

We had this problem once or twice with our trypsin. As others suggested maybe try a fresh bottle of your solution. We tend to aliquot and freeze ours if we are not to use the whole bottle very soon as it would go off.

However, what i tend to do for stubborn , non-detaching cells, I wash them first twice with versene solution or 50% versene/HEPES saline and then trypsinise as usual 2-4 mins should be sufficient.

good luck

Thanks for all the answers. But I am always using fresh trypsin as well and it does not explain why even TrypLE does not work. I have attached a photo of cells which were in culture for three weeks. I dont know what the problem is but maybe it is indeed the production of calcium or something like that due to differentiation. 

Hello again,

does your trypsin work on other cells? it could be a batch problem?

however, I would recommend you try the washes i suggested earlier and incubate the trypsin step 2-4min at 37C if you are not doing this already.

Based on my expriences, Trypsin is enough for mouse MSCs. If you have problems, maybe the cells were differentiated. I think the cells which could be deattached by Trypsin are MSCs. Campare these cells to the ones could not be deattached by RT-PCR, you  may find out the reason.

Yes I am also working with breast cancer cell lines and I havent experience any problem so far with the trypsin I am using. I am using calcium/magnesium free PBS and yes I am also doing the trypsin incubation at 37C. I will try your recommendations and update here if they work. Thanks a lot!

Hello Nese Erdem

Last year I experienced the same problem with my FVB mice BM derived MSC. I inferred that I took to long to passage the cells (more than 3 days) and they ended up with too much extracellular matrix for the enzyme to digest, maybe due to fybroblast differentiation. I could not characterize them, so I can not affirm that that is what happened. I started again and seeded more cells after each passage (1x10e4 cells/cm2), so they got confluent (about 80%) faster and I detached them more frequently (every 2 or 3 days) and the culture turned out ok. I could easily detach them after 3 minutes in 37°C TrypLE every passage and had good results in phenotype characterization and differentiation tests. 

Just saw your picture.  Thank you for uploading.  More than half do not have a great morphology.  They appear as old or activated MSCs.  They have lost their young undifferentiated morphology.  Is this picture towards the end of 3wks of primary culture P0?  3wks is far too long; it shouldn't take more than 10days, and thereafter should be every ~4days.  MSCs have a 20-30hr doubling time in culture.  My next hypothesis is your initial plating density is too low.  Try increasing as a very low seeding density may have stressed them out. 

Hello Nese

It would be great if you could elaborate a bit on the protocol u have used for isolation of BMSCs.

Im assuming that you have just flushed the marrow and seeded the cells on TCPS. I did that some time back and had a tough time isolating cells by trypsinization, nothing seemed to work. Trust me you are not alone in this situation, me and several people in my department had that problem.

I feel its an extremely heterogenous population that we get by this method. Later on we came to know that it contains a significant population of macrophages which attach to dishes like crazy. It is these cells which are stubborn and refuse to detach from dishes.

After this I have cultured MSCs from human bone marrow and adipose derived from goat. I havent faced any problems with them they grow really well.

I hope this helps you.

Your likely problem is that the cultures are allowed to reach high density, which promotes a large amount of ECM deposition as well as cell-to-cell contact and adhesion.  Once the culture density becomes high the cells will be virtually impossible to detach, and if they become detached, they will clump together and make it impossible to generate a single cell suspension.  Splitting cultures before they reach >70% confluence is critical.  We routinely employ gentle scraping in conjunction with trypsin, and as long as you let cells recover, e.g. 30 min at 4 degrees, they should be fine for flow.  

Dear Nese,

Most stem cells adhere to their substrate using two binding sites, a calcium dependent binding site and a RGD binding site.  You can release the calcium dependent binding site by first rinsing plates with Calcium/magnesium free PBS, and then follow up with a rinse of Calcium/magnesium-free PBS including EDTA or EGTA. A few minutes should be sufficient and you will see the cells rounding up.  I would suggest using collagenase (Worthington Biochemical) rather than trypsin to release cells at their the RGD binding site. Trypsin cleaves at lysine - arginine residues, which, unfortunately, are also found in cell plasma membranes.  Therefore, one could be damaging their cells with trypsin in the release process. We use collagenase for cell release and have gotten better than 98% recovery of viable cells using the above protocol: rinse with PBS (removes serum proteins), followed by rinse with calcium/magnesium-free PBS (removes exogenous calcium from cells), followed by rinse with calcium/magnesium-free PBS with EDTA or EGTA (we have used both) (releases calcium from the calcium-dependent binding sites and cells round up, except for specific RGD points of attachment), followed by collagenase digest (degrades collagen at RGD binding sites, while cell attachment filopodia remain intact). Depending on specific cell type (i.e., differentiated cells, progenitor cells, germ layer lineage stem cells, pluripotent stem cells or totipotent stem cells), our release procedures have lasted from less than 30 sec to as long as five minutes.

Dear collegues, may be anyone worked with reptilian cells. we need any recommendation how to remove the cells from the plastic. We make metaphase plates from them but trypsinization 0.05-0.25% gives not good results. The cells cultures from the lungs...

Hi Victor,

I have not worked with reptilian cells (though I would like to, sounds interesting), but fish cells and intricate mammalian cells can be detached using collagenase, either NB8 from Serva (now Nordmark!) which is low on tryptic activity, so you can leave it on the cells for longer, e.g. 1h, optionally followed by a quick TryplEXpress treatment (Gibco).

Alternatively, NB4 from Nordmark or Collagenase IV from Life Science might do the trick (then without subsequent trypsin treatment).

When cells really kept adhered I sometimes used cell scrapers after 5min trypsin treatment, and they lived happily ever after.

best regards,

Daniel

Hi,

I am also working with murine BM/MSCs and I also had a problem in de-attached cells. What I am doing is that I wash the plate twice with PBS and then I add 1 ml of Trypsin (EDTA, 0.02). This reduce the time of incubation. But I also found that if the cells are too old (around 2-3 weeks after harvesting), it took more time to be de-attached. Using collagenase, is also another way to de-attach the cells.