Osmolarity of external solution and growth media

Recorder

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Nov 16, 2015
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#1

Thank you for your attention. I have a question about the osmolarity of growth media for embryonic neuronal cell culture.





I saw in literature that the osmolarity of typical neurobasal/B27 growth media is about 235 mOsm. And so was my own measuring. When I measured the osmolarity of my neurobasal/B27 growth media, it was about 225 mOsm.





But, for the external solution, most people recommend about 300 mOsm external solution. Isn’t there any osmolarity shock when the cells are transferred to external solution for recording? I can’t understand why people don’t use 235 mOsm external solution.





Is there anyone who knows the reason?





Sorry for my awful English.
 

TheFFM

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Nov 16, 2015
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#2
It is probably just because the osmolarity of many common media are in ithe 296 mOsm/kg range for cells commonly patched like HEK 293, CHO and COS cells.



I think your suggestion to try working with ECF at an osmolarity that matches the culture media in which the cells were grown is sensible and you should investigate if you can make such a solution with the media recipie that you standardly use [NB thismay not be possible without diluting the desired concentration of some of the major ions included in your ECF recipie] . Alternatively you may consider adding some sucrose/mannitol to you culture media to increase it up to the 296-300 mOsm range.



Just to check - did you measure the osmolarity of your media with all the sera etc included or just the basal medium? Measure it with everything included.
 

Suola

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Nov 16, 2015
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#3
Recorder,



Different neuronal channels are selectively inhibited or sensitized by osmolarity of the medium. If you are doing patch clamp type experiments, this can be convenient way of inhibiting some channels that might otherwise interfere.... see example here. The time it takes for the osmotic shock to kill the cells is longer than it takes for you to finish your recording...



Or! Another potential reason might be that the osmolarity of your medium gets higher from the 235 mOsm (theoretical value, or measured outside the incubator), when you transfer your cells into the CO2 incubator because the CO2 dissolves in your medium and reacts with water to form bicarbonate... The bicarbonate adds to the osmolarity and the actual osmolarity of the medium might might then be around 300 mOsm. Maybe you can do the math and see if this is true, or use osmometer, if you have one....



Cheers,

 

Simon8

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Nov 16, 2015
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#4
Hi!



I also asked myself this question:

www.scientistsolutions.com/t19935-primary+neuronal+culture+osmolarity.html



My NB with all supplements and over night in the incubator has an osmolality of 223 mOsm/kg (our Osmometer uses superfreezing and gives only values per kg), wich is much lower than I expected.

If you believe Intivtrogen, this is an optimized osmolarity for culturing prenatal neurons. Otherwise they could have added some more NaCl, as Na looks to be pretty low in the media formulation (around 80 mM). So I dont think, increasing the osmolarity during culture is a very good idea (depending on the optimization statement from Invitrogen). But one has to try maybe.

But we should also keep in mind, that osmolarity increases during cultivation of our cells, unless you exchange the media completely and regularly (what at least my cells do not really seem to like). Or you compensate by adding water. But this is of course not the question here.



Of course, one has difficulties getting such low values with common extracellular solutions, especially when you add glucose.



So maybe one should also lower the NaCl in the patch solutions? What can happen? I guess Na is not as critical as K, but what about Cl?

I will try some things, and further think about this "problem".


 

Chemmjr

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Nov 16, 2015
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#5

In my organotypic slices I also use the NB-A+B27+Q combination. The osomolarity will increase in the incubator at least by two days. I don’t have my notebook but I have preformed some experiments (imaging, whole cell, fields) where I increased the osomolarity if my initial NB-A to 300 and have resulted in much higher osomolarity than recommended (iirc 360~370) after two days.



If you fear that the 'osomalarity 'shock' will negatively affects your slices you can, (albeit expensive) record in your NB-A media.
 

Simon8

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Nov 16, 2015
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#6
NB-A already has 17mM more NaCl inside (this is the only difference to NB). So what did the slices look like? They were the same as in lower osmolarity or more dead cells inside? I managed to have organotypic slices in normal NB (+supplements).



But now I am talking about dissociated cultures.

Anyways I wanted to record in media, because I want to check the cells firing in their media. Only thing is, you have to hurry with the recording, because it is not well buffered at air and I do not have an incubation unit at my rig.



I am not really fearing of shocking my cells, because my protocols were used before in our lab. But I rather ask myself now, if cells look better, when the increasing osmolarity is compensated (I need not so very young cells)?

And of course, I alsways think of ways to improve my protocols!
 

Wolf4441

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Nov 16, 2015
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#7
Hi!



I was just wondering how you test the osmolarity of the culture medium? I am a student and need to be able to test a modified cuture medium for IVF and cannot find any suitable protocols.



Best wishes,

Dani