what affects cell culture viabilty? - (May/21/2007 )
i have a question about viability of the cells , i mean if the cells loods healthy and bright .....what affects this?
my cells are 293 parental cells and i am following the protocol exactly for passing the cells or transfection but sometimes i noticed that my cells is not as healthy as others in the lab?
someone said may be trypsinization is the cause ? so i will tell you what i am doing and think with me please...
if i am using 10 cm dish i usually do 2 cell density 5x105 cells/dish and 2.5x105 cells /dish and do passage after 2 days so the cell confluency is always 60 or 70 %
wash once with pbs 5 ml aspirate quickly
trypsin-edta1% 2 ml for 1-2 minutes then aspirate
centrifuge at 300g for 5 minutes
then aspirate media , click the cell pellet by fingers add media 2 ml the pipet up and down 5 times to get a single cell suspension
then count and always my cells is very goon in trypan blue *i mean no blue cells*
seed
but when i took from this cell to do transfection or second passage my mentor said that cells dont look healty enough?
how to tell?
where is the problem?
thankx
There could be a lot of reasons for this.
The pH of your media is important, make sure your CO2 level is in line with the composition of the cell medium, there are differences between 5%, and 10% CO2.
Make sure that the cells get enough to "eat", consider the glucose level in the media as well as the serum percentage.
Consider the attachment, some cells only grow on "coated" surfaces such as fibronectin, collagen, laminin etc.
Also check the pinned topic of contamination here.
Cells left in the same media for a long time (or short time if they are numerous or hungry) deplete the energy sources quickly and also produce waste products (some of my cells required media changes every day).
You trypsinization seems to be working but I just want to stress that serum inhibits trypsin and a quick serumwash is a good way to stop the digestion after subculturing.
Hope this helps! 
D
my cells are 293 parental cells and i am following the protocol exactly for passing the cells or transfection but sometimes i noticed that my cells is not as healthy as others in the lab?
someone said may be trypsinization is the cause ? so i will tell you what i am doing and think with me please...
if i am using 10 cm dish i usually do 2 cell density 5x105 cells/dish and 2.5x105 cells /dish and do passage after 2 days so the cell confluency is always 60 or 70 %
wash once with pbs 5 ml aspirate quickly
trypsin-edta1% 2 ml for 1-2 minutes then aspirate
centrifuge at 300g for 5 minutes
then aspirate media , click the cell pellet by fingers add media 2 ml the pipet up and down 5 times to get a single cell suspension
then count and always my cells is very goon in trypan blue *i mean no blue cells*
seed
but when i took from this cell to do transfection or second passage my mentor said that cells dont look healty enough?
how to tell?
where is the problem?
thankx
the number of passage could be too high; go back to a lower passage number; use recombinant trypsin such as TrypLE; as suggested, it could be some reasons...
my cells are 293 parental cells and i am following the protocol exactly for passing the cells or transfection but sometimes i noticed that my cells is not as healthy as others in the lab?
someone said may be trypsinization is the cause ? so i will tell you what i am doing and think with me please...
if i am using 10 cm dish i usually do 2 cell density 5x105 cells/dish and 2.5x105 cells /dish and do passage after 2 days so the cell confluency is always 60 or 70 %
wash once with pbs 5 ml aspirate quickly
trypsin-edta1% 2 ml for 1-2 minutes then aspirate
centrifuge at 300g for 5 minutes
then aspirate media , click the cell pellet by fingers add media 2 ml the pipet up and down 5 times to get a single cell suspension
then count and always my cells is very goon in trypan blue *i mean no blue cells*
seed
but when i took from this cell to do transfection or second passage my mentor said that cells dont look healty enough?
how to tell?
where is the problem?
thankx
Dear Spanishflower,
Just a couple of comments:
i) Trypsin concentration should be 0.25%. If it is warmed then 1 minute is fine.
ii) Centrifuging causes damage to cells so use the minimum speed. I use 100g which is sufficient to pellet viable cells.
iii) Trypan blue is an EXCLUSION DYE and is very INSENSITIVE. Thus you will get 99% viability BUT you will have a greater percentage of cells that potentially are apoptotic but which will STILL EXCLUDE THE DYE.
iv) FCS/FBS is the single most important additive to the medium. ALWAYS use quality serum....New Zealand origin being the best(£250/500ml), British serum being the worst (£30/500ml).....YOU GET WHAT YOU PAY FOR.
v) As said before CHECK CO2 levels in the Incubator (Fyrite), keep the incubator HUMIDIFIED.
vi) REGULARLY check for MYCOPLASMA CONTAMINATION.
vii) Check out the different Tissue Culture plastics i.e. OPTIMISE your growth conditions.
Hope this is useful, tissue culture is easy BUT you have to obey the rules........30 years experience helps as well.
P.S. Your mentor should be helping you with this.
thankx all for your reply
but may be i didnt make myself clear enough
we share incubator, trypsin-edta 1%, media , coated dishes....and every thing in the lab ( i dont mean by share to use the same bottle , i mean we do use the same brand of everything) and everybody is happy by their cells....
so it must be me that is doing something wrong?
about centrifugation i tried 100 g as you told me before Rhombus but i didnt get any pellet while my collegues here centrifuge at 500 g and more and their cells(293A) are ok?
i watched one person doing her passage today and she left her cells 3 minutes in trypsin and did 11 times pippeting up and down and her cells are ok? for me i never left my cells in trypsin this time or did this number of pippeting....
going crazy really here ....today after transfecting my cells i got empty spaces in between them? its not because of an error in aspirating or adding media , my mentor said its because my initial cell condition was not good>?...
Well, did your mentor explain in what way the cells look bad? It is a very fuzzy definition. If I were you I would thaw up a new vial from the freezer and start over, throw away all old media bottles, maybe even ask for an aliquote from your labmates.
Good luck
P.S. I completely agree with the comment about your mentor!
Thankx DLY
i took some cells from a post doctor here and i did passage for them yesterday and they are fine today thank GOD .
i asked one girl in the lab to show me her transfected cells today*she did transfection for her cells at the same time as mine* and she also got the same problem of having many gaps in between cells despite 90% confluency at the day of seeding.... so its not just me ...
need more practice for good seeding in 12 well plate.
No worries, happy to help! I also get "holes" in my cultures sometimes, it depends on how fast the cells crawl over the surface and how fast they divide I guess. To fast division/migration makes the cells grow in islets...
but may be i didnt make myself clear enough
we share incubator, trypsin-edta 1%, media , coated dishes....and every thing in the lab ( i dont mean by share to use the same bottle , i mean we do use the same brand of everything) and everybody is happy by their cells....
so it must be me that is doing something wrong?
about centrifugation i tried 100 g as you told me before Rhombus but i didnt get any pellet while my collegues here centrifuge at 500 g and more and their cells(293A) are ok?
i watched one person doing her passage today and she left her cells 3 minutes in trypsin and did 11 times pippeting up and down and her cells are ok? for me i never left my cells in trypsin this time or did this number of pippeting....
going crazy really here ....today after transfecting my cells i got empty spaces in between them? its not because of an error in aspirating or adding media , my mentor said its because my initial cell condition was not good>?...
I have no problem in trypsinizing my cells and how many times pipetting up and down. they still look OK.
But when i do trypsinization, i observe the cells under microscope few times after i put in trypsin. and when the cells morphology start to change (become rounded and looks like going to detach), then i put in warmed media with FBS (mix into the suspension with trypsin) to stop the trypsin's reaction and resuspend. spin down cells and discard media.