subculture primary renal tubular epithelial cell - (Apr/09/2006 )

Hello,
I have cultured primary renal tubular epithelial cells from mouse for months.But I can not subculture it . The problem is the primary cells are adherent to the vessel,but after I subculture the cells with trypsin(1:250),0.2%.the cells can not adhere to the vessel.They become round,suspended.The meidum that I use is PRMI1640(invitrogen),15%FBS,antibiocs.I am puzzled,and do not know what I should do. Can you help me ?

This might not help. Just wondering how long did you trypsinize you cell? Is your cell sensitive to trypsin? And did you do PBS wash to remove FCS before adding trypsin?

hi wing ! just reduce the time of trypsinization to 5-6 min. donot wait for the cells to disadhere but gently tap the flask against the floor of the laminar hood so as to remove max. no. of cells. you said the cells are loosing viability after disadhering them by trypsinization. complete inactivation or removal of trypsin is necessary for some cells which are trypsin sensitive. either wash the cells properly or use higher amounts of FBS.
also seed optimum no. of cells while you are subculturing
all the best

hi wing ! just reduce the time of trypsinization to 5-6 min. donot wait for the cells to disadhere but gently tap the flask against the floor of the laminar hood so as to remove max. no. of cells. you said the cells are loosing viability after disadhering them by trypsinization. complete inactivation or removal of trypsin is necessary for some cells which are trypsin sensitive. either wash the cells properly or use higher amounts of FBS.
also seed optimum no. of cells while you are subculturing
all the best

If your cells are still rounded and not attaching then they are either dead/dying or the trypsin hasn't been sufficiently inactivated.

If you preheat your trypsin to 37C your cells should lift in no more than a few minutes. Make sure ALL the media has been removed and give the cell surface a good rinse with PBS w/o Ca & Mg and remove it completely prior to adding the tryspin. You can do two washes if you like but generally one is fine so long as all the medium has been removed prior. How much trypsin do you add? You should try to minimize the volume. One way is to add enough to so that the entire cell surface is covered (roll it around the vessel) and then tilt the vessel and remove the majority of the excess trypsin. I leave about 0.5ml in a 25cm2 culture vessel, but some in my lab would leave more, others a little less.

Incubate the vessel after adding the trypsin as this will help speed its action and make your cells lift more quickly. Check it after a few minutes under a phase microscope and if the cells are beginning to look rounded give them a tap on the bench as Shiva has suggested. We are sometimes quite brutal with how hard we tap but it would depend on the cell line. If the majority of cells lift from the surface you're done. Otherwise return the flask to the incubator for another minute or two.

15% FCS should be heaps to neutralize a small amount of trypsin. Break up any clumps of cells by genetly pipetting up and down (again we can be extremely brutal with this - doesn't seem to bother our cells lines). I have mainly grown human fetal cells and we have never washed cells following subculture but if your cells really are sensitive to trypsin than this is probably the best option if nothing else works.

Good luck.

[quote name='karyotyper' date='Apr 11 2006, 07:08 AM' post='47670']
hello,
Thanks for you advice!Yes,I have removed all the media and give the cell surface a rinse with D-Hanks w/o Ca & Mg for twice and remove it completely prior to adding the tryspin.Does it matter using D-Hanks or PBS to rinse the cell surface? I used about 2ml 0.2%trypsin in D-Hanks in a 25cm2 culture vessel. Maybe it is too more.
Best Wishes!

i think u should post ur culture protocol here. maybe sth wrong in your protocol.