Hi Madhuaram,

I have had sometimes similar problems with cells which express high amounts of keratin. First I washed the cells 2x with PBS, then I removed the trypsin solution after 5 min, then put a new trypsin solution on the cells for another 5 min. After that I still had to shake the flasks vigorously.

We also observed that the cell density had an effect, confluent cells attached stronger than non- or near-confluent cells, so I tried not to use cells with more than 80% confluence.

Best regards,

Carsten

Hi Madhuaram,

I have had sometimes similar problems with cells which express high amounts of keratin. First I washed the cells 2x with PBS, then I removed the trypsin solution after 5 min, then put a new trypsin solution on the cells for another 5 min. After that I still had to shake the flasks vigorously.

We also observed that the cell density had an effect, confluent cells attached stronger than non- or near-confluent cells, so I tried not to use cells with more than 80% confluence.

Best regards,

Carsten

Dear Madhuaram,

I agreed with Carsten suggestions. Just to add 2 more points

1) don't grow cells too confluent as that will hamper the effect of trypsin, so may be try to see less cells but in more flask (around 80% confluency should be fine)

2) you can change the concentration of trypsin as well or try another enzyme mix

All the best

Tushar

Dear Madhuaram,

I am agree with Carsten Schmidt and Tushar Tomar that the cells confluency should be 80%. You also can try to little increase the volume of trypsin solution..

Thank you for your suggestions. But my cells are taking too much time in trypsinization,  hope so longer incubation with trypsin not going to affect my cells property. Cells on which i am working they are slow growing and having property to grow in clumps so although cells are not fully confluent but as they grow in clumps they have tight interaction. And what is the maximum conc. of trypsin i can use for trypsinization.

Dear Madhuram,

I am afraid if you can increase the concentration of trypsin (0.25%) with such long time incubation (>15 mins). My personal suggestions would be

1) Either combine trypsin with collagenase: this is mainly for high-density cells or cells that make multilayer formation in clumps (don't know if that's the case with your cells)

2) Try commercial products as substitution of trypsin with equal potency and don't affect cells like 

TrypLE™ Express or  TrypLE™ Select 

https://www.thermofisher.com/nl/en/home/life-science/cell-culture/mammalian-cell-culture/reagents/trypsin/tryple-express.html

All the best 

Tushar

Does your trypsine work well on other cell, an easier one, like HeLa etc? It might be decomposited already and loss its activity. TrypLE is a nice try, and stabile at room temp, but demands extra work on it, so watch its description.

Anyway, there are cell lines could hardly split by regular way. MDCK is a nice example.

Dear Madhuram,

I agree with Tushar. Prolonged incubation with increased conc. of Trypsin may harm your cells. For such a difficult cells we used 'Accutase' from Sigma. It worked well.

Best,

Kamil

Dear Attila we are also working on other adherent cell lines and trypsin worked well with those cells, So in that case probability of decomposition of trypsin is negligible.

Did you always have this problem with this cell line or did it just occur recently, also could it be maybe related to this particular batch of trypsin that you are using? Also at what temperature are you incubating the trypsinized cells?

Dear Madhuram,

I would suggest that following the two washings (it is very important to eliminate all trace of FBS) you add your trypsin to the cells and incubate at 37°C for 5 to 10 min. You need however to monitor closely the cells.

Another possibility, would be to incubate your cells after the PBS washing and before  the trypsin for 2 min with some Versene solution. This protocol was used for trypsinization of a very particular mouse cell line growing in 3D, the MIN6 cells.

I would also like to add a comment: there is no universal protocol for trypsinization. I mean you cannot use the very same protocol for all cell lines or for all primary cultures. You need to adapt every time your protocol for a given cell line and even (it happens but no so often) for different clones of a particular cell line.

I hope that this post will help you.

Regards,

Smaragda

Thanks for your suggestion . Thanks to all