5 replies to this topic

[CMIRC]

Hello all.

I am a newcomer to this blog. Would like to congratulate the founders and the members of this blog and website as a whole as I have found solutions to many of the problems In the lab.

I am culturing PC3M cells (Highly metastatic cell line of prostate cancer) and am having problems in terms of sub-culturing. Does anyone have any experience in culturing PC3M cells.

Please help!!!

[Kat T]

Hey there,

I culture a GM version of the PC3 cell line.  I'm not quite sure what your problem is with sub-culturing.  Are you wondering when to subculture, or are you having issues with the cells after they are sub-cultured, or are you wondering which way to subculture them?

If you are having troubles getting them to grow, have you checked for mycoplasma contamination or a background fungal/bacterial contamination (particularly if you add antibiotics to your media)?

I have found that the PC3 cells have to be grown in a larger than usual flask (I use a 125cm² flask, compared to the usual 80cm² flask) as the cells seem to be quite disperse, and appear to inhibit each others growth when they become too confluent.  My protocol for subculturing is as follows:

1) select a fairly confluent flask (about 75-80% coverage)
2) rinse the flask with PBS (about 20ml for a 125cm² flask)
3) discard the PBS rinse
4) add 2ml 5% trypsin and put flask back in the humidified incubator (95% humidity, 5% CO₂ at 37 degrees C) and wait about 2min until the cells detach
5) Add 10mL of media and split the cells as required (generally once the flask is quite covered I split the cells three time a week; Monday (one in three), Wednesday (one in two), Friday (one in three)
6) make the volume in the flask back up to 50mL media (for the 125cm² flask)

I grow my cells in F12 media with 10% FBS.

Hope this helps.  Good luck!  

[CMIRC]

Thank you very much for the reply. It was very informative and helpful. I follow a similar way of sub culturing to yours apart from the size of the flask ( I use T75), and after trypsinisation I centrifuge the cells at 200 x g for 5 min to get rid of trypsin EDTA. I looked around online for protocols to culture PC3M cells but could not find any. I did find protocols for PC3 and realised that non of them involved centrifugation after detaching the cells using trypsin. Is there a reason for that?

secondly problem I have with sub culturing is that the cells become very stressed and then do not recover or remain in a stressed state even after splitting. I can not seem to figure out why this is happening.

Thirdly is it ideal to trypsinise PC3M cells or any other adherent cell line two days in a row meaning if I trypsinise cells on monday to split, can I trypsinise them again the next day either to split or for experiment?

[Kat T]

It sounds like your method of splitting them is stressing them unduly.  this could be a number of reasons, but the first ones that spring into my head are

1) the cells have become too cold during the passage (do you heat all of your reagents up before using them and approximately how long does it take you to split them?)
2) you have been rough with them (i.e. pipetting them up and down after centrifuging can do quite a bit of damage to them)

The PC3 cells are what I would consider 'sensitive' cells, in that they do tend to sulk if not treated nicely.  Have you done work with other cell lines, or are these your only ones?  Do you centrifuge your cells in the trypsin, or do you add media containing FBS to them prior to centrifugation.  You really don't need to worry about removing the trypsin, just so long as you media contains some sort of protein (i.e. your FBS) as the trypsin will become deactivated by the huge amount of protein present in the media.  The only time I centrifuge my cells is when I first initiate them to remove the DMSO as this inhibits their growth.

I wouldn't trypsinise two days in a row.  Try to organise your trypsinising so that they have at least one recovery day.  Remember that you don't have to split at the same ratio each time, so if you don't plan to use them for 4-5 days you could always split them harshly and do a media change halfway through (i.e. just pour off the media and put new stuff in).

Hope this helps

[CMIRC]

Thank you Kat T for the fast response. I think both the reasons mentioned above for stress apply to me somehow. I do not warm the media or the PBS however I do leave them to come to room temperature before using them. However must agree at times I have used cold PBS to wash and cold medium to grow the cells. Thank you for that suggestion. I will implement this in culturing these cells.

Secondly I do not pipette majority of the times, however I do centrifuge the cells post trypsinisation atleast twice if not three times and I think this may be stressing the cells out. So thank you again for that suggestion.

I am not new to cell culturing . I have cultured other cell lines like THP1, Jurkat, HL60, and skeletal muscle, However the first three are non adherent and the last one is a primary cell line which I take care off very carefully following strict culturing protocols. However assuming PC3M cells to be more rugged as they are difficult   to kill, I probably did not pay attention to culturing these cells more carefully. Thank you once again for your suggestions and will definitely implement them in my cell culturing technique.

One more question, would you happen to know of a way of inducing early apoptosis in these cells without using chemical agents? sorry for being out of context.

[Kat T]

Hi CMIRC

Sorry no idea about the apoptosis.  I use them to measure androgen levels in samples.