3 ways I guess you may want to consider:

1- use calcium/magnesium free PBS like Patrick said;

2- use higher EDTA concentration with the trypsin or alternatively incubate your cells for 10 minutes with 0.05% EDTA then trypsinize;

3- have the trypsin from the 37C waterbacth immediately to the flask and then put the flask in the incubator, this warm environment will help detach them without serious effect on them.

Hope that helps :).

Did you try to rinse the monolayer with Ca2+/Mg2+-free PBS before adding the trypsin solution? It helps removing divalent cations from adhesion proteins like cadherins, which is sufficient for some cell lines to remove them from the culture vessel without further trypsin treatment. Furthermore, trypsin inhibitors possibly added from serum supplementation are removed by rinsing.

3 ways I guess you may want to consider:

1- use calcium/magnesium free PBS like Patrick said;

2- use higher EDTA concentration with the trypsin or alternatively incubate your cells for 10 minutes with 0.05% EDTA then trypsinize;

3- have the trypsin from the 37C waterbacth immediately to the flask and then put the flask in the incubator, this warm environment will help detach them without serious effect on them.

Hope that helps :).

What the others have said with some reservations: Trypsin degrades itself, and therefore the potency declines with time. Its best not to warm the whole bottle more than a few times. So aliquot and freeze when you get a new bottle. You can also use TrypLE ( http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Culture/Mammalian-Cell-Culture/reagents/Trypsin/TrypLE-Express.html ) that is a little more stable.

With human MSC I wash with warm ca/mg free pbs, then incubate in ca/mg free pbs for a few minutes before trypsin. I also smack the dish a bit... but this can be bad for some cell types.

Adding to others,, might not be much helpful but this was what I observed lately!

-If the cell population is highly dense that usual, they take longer times to detach which is not good.. So try to not leave them for many days than its regular sub culturing times!

-Also, After adding trypsin try placing them in incubator, use warm media, w,arm trypsin might help ,, Make sure you remove all media from plate and wash with PBS as serum hinders action of trypsin on cells!

-Another one is check if you need to increase Trypsin concentration to 0.25% and check if the added volume of trypsin is covering entire plate (usually for 100mm petri dish,

I add 1 mL of 0.25% trypsin but some use 2 mL or a mL of 0.5% sometimes)

About floating after sub culturing, is it floating like that after an hour too.. I mean did you check if they are adhering to the plate after an hour of incubation compared to immediately after seeding them? That made difference in my cell lines as it took time to adhere actually. And when I used bone cells I observed that they needed collagen coating to resolve this kinds of problems.. so check if any coating works!

Hope at least one of the points I made help resolve your problems. Good luck!

they are no longer msc, you changed them or started some pathway leading to death. the best way to detach them is to use trypsin/edta (invitrogen) and before you put trypsin in the flask do the last washing with a ml of trypsin, suck it up and add the amount of trypsin you use to detach the cells. incubate @ 37 for no more than 3/4 min and then when out tap flask gently at the side. when you put dmem in to inactivate trypsin remember to let it flow on the side where cells are attached, this will finish detaching them.

I do the same process as mentioned by using Ca and Mg free PBS for the washing of the cells prior to trypsinization .

But one thing I am doing wrong is that I do completely make the vial to thaw at 37 C by keeping it in 37C incubator (I have a shaking incubator for thaw of vials and FBS) and then use it for the process. And this process is repeated every time I do trypsinization. One more thing I do not keep the vial in freezer but do place in refrigerator.

As quoted by William Rice, I do smack the flask and plates to and fro so to increase the detachment process, but irrespective of smacking or aspiration or addition of Trypsin using wide tip pipettes the cells detach in less number.

Can i use a higher amount of trypsin concentration i.e. o.5% as mentioned by Rt P, bcos with increasing con. I would have to lessen the incubation time as if i keep the cells in trypsin soln for a longer time the adherens protein present in cells would degrade and the cells would be unable to adhere completely for further studies.

The cells very much remain non adherent after trypsinization, as after 4 days the cells are still floating.

Meera- overtrypsinizing will kill your cells, so proceed with caution. If you are working with primary bone marrow aspirates, you should expect to recover a number of adherent cells such as monocytes that are not MSCs. It could be that your cells that fail to trypsinize are not MSCs but another subpopulation of cells, in which case, you shouldn't worry about their lack of detachment, since they are not your target population.

Most MSCs produce large amounts of collagen. It is a bit more expensive, but digestion with collagenases, following by a very short trypsinization step, often yields better results (more cells released with less cell death) than using trypsin alone.

Don't try to shear your partially trypsinized cells off the dish using culture medium. Most of these cells will simply rupture and die, so this is not worth the effort. A few strong taps of the flask is really the best approach for mechanical agitation.

Increase of concentration should be correlated to volume you add and working area. For instance, for the epithelial cells, I use 1 mL of 0.25% on 100 mm Petridish. However, a friend of mine uses 0.5% of 2mL for flask. So basically it depends on the working surface. I was wondering if adding trypsin is washing any adherent proteins! Here is a paper which might help!

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

For monocytes/macrophages, trypsinizing adherent colonies was never sufficient- we always had to use cell-lifters. Note there is a difference in the bevel angle between cell "lifters" and "scrapers", as using the latter will like shear the membranes of most of your population. As Daniel noted, over-trypsinizing the cells is cytotoxic, which could explain why the "cells which float" cannot be subcultured. If the EDTA-trypsin that others suggested doesn't work, try either using cell lifters, or digest the adhesion plaques with Accutase (from ThermoFisher: http://www.thermo.com.cn/Resources/200802/productPDF_26369.pdf): it's much less cytotoxic to your cells, and I've been able to digest not only cells from plastic, but soft-agar colonies out of agar and into single-cell suspensions. It works quite well, with no detectable cytotoxicity (to our cells) after 15min of incubation. As with Trypsin, make numerous aliquots of the Accutase solution, to minimize freeze/thaw cycles.

I found if the cells become confluent, there are difficult to detach from the culture surface. In that case, I always add trypsin twice.

When epithelial cells (or several other cell types) become confluent, the increased adhesion that you may observe results from connexin or related cell-cell junctions, and not necessarily increased cell-surface adhesions. These connexin-type junctions typically require divalent cations to form. While EDTA treatment does not necessarily "strip" Ca/Mg from the connexion complexes formed by adjacent cells (this has been published in the context of wound-healing assays), it does mean that these junctions will disassociate rapidly upon physical trauma, such as a pipette-tip induced "wound", or physical agitation (i.e. tapping and swirling), and the EDTA will prevent these junctions from re-forming. Bottom line, cell-cell and cell-plastic interactions result from different structural components, and thus can be disrupted in different ways.

Dear Meera,

I will prefer to use cell scraper to detach cells.

We are using cell scraper for strongly adherent cell line.

Thanks

I would also check the quality of the T25 flasks.

Hi Meera,

according to the posted site, 10 minutes trypsinization time is the norm. How long it will take depends on how fast your flask warms up. As suggested by previous comments, add warmed up trypsin. Then place flask into incubator for about three minutes, then remove it and quickly tap the sides. If the cells are just starting to come off, put it back for a further 1-2 minutes and repeat the process. How fast the cells come off depends on how fast you work. If you are too slow when visualising, trypsin cools down and activity reduces. However, the cells that came off would be still affected and then they may not attach upon seeding.

One very important thing. When about 90 percent of the cells detached, quickly add the neutralising solution that stops the activity of trypsin. For most cells it is FCS, I am not sure what is right for your system. This serum then protects cell integrity and also helps them to attach. The process is a bit of trial and error. Occasionally one can use a pipette to detach the last 15% of attached cells but I probably would not worry about these cells as then the rest of the detached cells may be affected.

http://www.rndsystems.com/literature_ccm004.aspx

Cheers

hi,

i have also isolated the mesenchymal stem cells from mouse bone marrow, but i never faced the trypsinization problem. infact my cells are very easy to trypsinize. i use simple trypsin/EDTA solution (0.025%), but never tried TPEG. Another thing which i would like to mention that trypsin always act only at 37 degree, so prewarming is quite essential.

you can also try doing a quick rinse with trypsin - like 5-10 seconds and then doing a real trypsinization event. I found that this helped greatly

As Kewalin mentioned, confluent MSCs can be relatively difficult to trypsinise when confluent. In those cases (and when working with keratinocytes, which can be particularly tricky to dissociate), I do something similar to what Mirza said: after rinsing with PBS, add trypsin, incubate for 1-2 mins until cells begin to round up but not detach; carefully remove the solution, and add fresh trypsin. Cells will almost instantly become round and detach. If this doesn't work, another round of fresh trypsin may be necessary.

Why I get a white clump after centrifuging the trypsinized mesenchymal stem cells?? is there a way to get rid of it?

quick question-- would trypsin LE be a better alternative for problems faced with trypsin EDTA? I found that trypsin LE is more gentle to the cells, especially of primary origin. Any thoughts on this?