I have seeded cells (MC3T3-E1) in 10 different 12-well plates and checked for attachment a couple of hours later. At this point most of the cells attached nicely. The day after, however, the cells seeded in the four corner wells were all dead, whereas the cells in the remaining wells were ok.
Is there an explanation for this?
Maybe to much or to less number of cells in the corners?
When to much the overgrowh, when to less they maybe haven't the cell contacts the need?
All wells have the same amount of cells from the same cell suspension. Given that the other wells are fine, I don't think the cell density is the problem.
I have heard anecdotally that this is due to the manufacturing process of plates and that you should avoid using the outer wells of plates. I'm not sure I hold much stock in it though.
maybe too little volume? it happened to me, when I used 24 well and I have little water in the incubator.....
Sorry Matthias, after your answer to my first posting I see that I missunderstood your question.
For 96-well plates I find these information: http://www.level.com.tw/html/ezcatfiles/vipweb20/img/img/20297/Corningcellculutretroubleshootinguide.pdf
See page 6 and then Evaporation.
I don't think that this effect is also high in 12 well plates where the volume of media is much higher. But this shows that not all wells are excactliy the same. So maybe the atmosphere in the corners differ because of Evaporation effects?
Matthias, edge-effect is what John refers to is often happening on multiwell plates due to the extra heating taken up by the edges of the plate when inside the incubator.
You can try an "home-made" solution by filling the void of the plate with a small amount of DI water or else look into/contact http://www.advancedmicroplates.com/. they will certainly resolve your issue if you are running large screening.
Furthermore MC3T3-E1 are very robust as cell line; I have worked with them and never had much trouble once you resolve the seeding density. I would then suggest to have a full temperature check of your incubator for each tray and location where you put your plates. The easiest is to inserting a 12-well plate with same amount of DI water + termocouples in all well. Have a 24hr record and check it.
Hope it will work for you. Keep me posted.
I think that the problem is due to the fact that the 4 corners of the cells are "less protected "(for example, having two exposed sides have greater exchange of air, the pH varies more and the temperature is colder). Excuse me for my english!!!!!
by Simonetta
Wouldn't parafilm around my plate prefend air exchange? This in itself might have a baleful influence on the cells, no?
Matthias I agree with you..if you put parafilm around the plate you will not have co2 exchange and so the medium will change pH. My suggestion..increase slightly the volume of medium in the corner well
@fulvio . Lo credevo anche io , ma ho letto che il parafilm favorisce gli scambi gassosi preservando le colture dalla disidratazione ,che credo sia proprio il problema di matthias.
@matthias.I also thought about this, but I read that the parafilm promotes gas exchange while preserving the cell culture from dehydration ,that I think is your problem
@Simonetta...you are right...a quick research on google shows me that parafilm has an high permeability for CO2..something I did not know...thanks for the info :)
Thanks for the info, I also don't know this.
"research on google" - funny formulation :-)
Dear Matthias and All, I would be careful over the excess use of parafilm. The problem with parafilm is the fact that is not sterile and electrostatically can carry airborne contamination into your wells. Furthermore to sell it at such a low cost it is a not pure mix of polymeric materials. I would encourage you to work on semi-permeable adhesive well plate covers which can prevent contamination and reduce evaporation. However I would still stand with my previous comment over edge-effect quantification and compensation.
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