Cell culturing tips - (Nov/03/2013 )

Greetings everybody.

This topic addresses the discussion of recommended surface area and volumes for cell culture vessels.

I am relatively new in the field of cell culturing and would really be thankful if anyone is willing to share their cell culturing tips/techniques in this thread.  I am constantly being bugged by the question of the volumes of cell culture reagents when I am doing cell passaging, seeding or even freezing.  Maybe I can start with cell passaging, followed by seeding and then freezing.

I am currently culturing H9c2 cells and just completed one passage.  Is it safe to say that there is no hard and fast rule when adding say, PBS, trypsin and culture medium?  

I started by thawing 3 ml of cells and adding 7 ml of culture medium on Day 1 (Fri), allowing it to grow over the weekend.  Day 4 (Mon) is when I will be doing cell passaging.  So basically, I would aspirate the 10 ml of cell suspension, wash with PBS (6 - 8 ml?) followed by the addition of (3 - 4 ml?) trypsin before neutralisation with (3 - 7 ml?) of culture medium before centrifuging in a Falcon tube and transferring to a new culture plate.  Before centrifuging, I would remove half the volume of cell suspension as I am doing a 1 : 2 passage.  The reason for the question marks is that I am uncertain about the preferred/recommended volumes of these reagents.

Does anyone have an idea of these volumes and also, if possible, please do share your cell culturing tips and how you get desired results for your cell culture work.  It would be really helpful if you could also post microscopic images of your cell culture work with labels and magnification.

Thank you!

The volumes you should use for growing the cells are dependent on the vessel area you are using for the culture.  The following are a general guide - 96 well plates 100-200 ul, 24 well plates 0.3-1.0 ml,  6 well plates 1-2 ml, t-25 flask 7-10 ml, t-75 flask 10-20 ml, t-175 25-50 ml.  Have a look at the documentation for your particular brand of culture vessel, many of the companies provide guides to this sort of thing.  A general guide is that you need about 5-10 mm of medium above the cells to allow adequate gas exchange.

Likewise the volumes you should use for trypsin are dependent on the vessel area. You need enough to just cover the surface if you tip the vessel.  For L24 I would use 200 ul, L6 0.5 ml, t25 1ml, t75 2 ml, t175 3ml.  The volume of neutralization is dependent on how much trypsin you have added, but 1-5 x trypsin volume is common. 

There are some rules, for example, try to minimize the exposure to trypsin, keep everything sterile, do not allow cells to reach confluency before you passage them (especially true for your myoblasts, because they might start fusing if they are too dense).  When defrosting cells, it's super important to do it as fast as possible. Never leave the vial in waterbath. There should still be a little ice left when you take it out.

After you first plate your frozen cells, it is best to change the medium as soon as the cells are attached, and definitely the next morning. It may not be a good idea to let them sit in it over the weekend.

If you grow your cells in a t25, 5-6 ml of medium and 0.5 ml of trypsyn is enough. If your cells are easy to detach, add 1-2 ml of trypsin and remove it immediately, leaving a thin film layer of it over the cells. If they are a little trickier to detach, add 0.5 ml of trypsin and let it sit on the cells, checking all the time. Have someone show you how to hit the flask with your hand to help dislodge the cells.

Often it's not necessary to centrifuge the cells after trypsinization (unless you need to count and resuspend to a certain concentration).  Check if your particular cells require this step by looking in the literature.  If this step is not specifically recommended, just transfer half of your cell suspension to the new flask. It's a good practice to leave the rest behind in the old flask (add some fresh media) in case something goes worng with your new flask, and also to educate yourself about what happens to the cells when they get old/confluent/etc.  It is a bad practice to use the cells from those old flasks, unless you absolutely have to.

Hope this helps. Feel free to ask more questions.