This might be a problem of osmolality (medium, ACSF and pipette)? Did you compare "used" medium with your ACSF? Depending on feeding schema we found (in 6-well multidishes) an increase of ~7% over 48 hours (due to evaporation). This might be even worse when changing only the half of the medium. We are using the Stoppini method either with serum or OptiMEM/Neurobasal B27 suppl. serum free. No problems with whole cell up to two weeks. (You might drop by next time we have cultures ;-)

We are regularly using the following medium for Stoppini-like hippocampal cultures and record from 2-3 weeks old cultures with no sealing problem. Medium is replaced every other day. Good luck.

Compound----------Gibco #----------Add (ml)

MEM-------------------21090-055-----25

HBSS 1X-------------14185045------12.5

Horse serum---------------------------12.5

Pen/Strep------------15140122------0.5

Glucose 1M-----------------------------0.8

Ascorbic acid 1mg/ml----------------0.1

HEPES 1M-------------------------------0.4

B27--------------------15140122------0.5

Check osmolality (310-320 mOsm)

Filtrate and refrigerate

Prepare weekly

There is a paper by De Simoni & Yu describing the steps pretty well (Nat Protocols, 2006). I my experience the classical 25% horse serum containing medium enables much longer culturing times than serum free media. With this we have patch clamped cells after months in cultures, with no difficulties or altered electrophysiological properties compared to younger cultures. We add a bit of sucrose (20 mM) though, as in Cronberg, Rytter, et al, Stroke, 2004, and it works very well. We cultured in 24 well dishes, and changed medium 3 times per week (so you can enjoy your weekend out of the lab).

I also follow De Simoni's protocol, and it's working pretty well for us.I culture them in 6 well plates, so changing half of the 1 ml media every 2nd or 3rd day works well.I use the cultures for calcium imaging and whole cell current clamp recording. In my experience placement of electrode while recording is important, as these cells are not as robust as acute slices and for the health of the slices level of media is very important. Dr. Tonnesen do you add sucrose just to balance osmolarity or it has some other significance? Can somebody tell me which part of the slices start deteriorating first, as in my cultures it is subiculum-cortex interface which gets disorganized after 2- weeks.

Did you tried change your pippet? Maybe if you change the size or the material it could helps.

There are several good references for hippocampal slice cultures for electrophysiology. However, it makes a difference if one is going to use a multielectrode array for recording or a standard recording chamber with just 2 or 3 electrodes. See these references for starters:

1) chapter 2.7 by Thompson and and Gahwiler, Organotypic Hippocampal Slice Cultures in the book called Practical Electrophysiology Methods, editors, Kettenmann and Grantyn

This might be a problem of osmolality (medium, ACSF and pipette)? Did you compare "used" medium with your ACSF? Depending on feeding schema we found (in 6-well multidishes) an increase of ~7% over 48 hours (due to evaporation). This might be even worse when changing only the half of the medium. We are using the Stoppini method either with serum or OptiMEM/Neurobasal B27 suppl. serum free. No problems with whole cell up to two weeks. (You might drop by next time we have cultures ;-)

Hi Benjamin, you may like to check out this video protocol available on JOVE:

http://www.jove.com/video/2462/organotypic-hippocampal-slice-cultures

This is from a very reputable slice physiologist I know at University of Washington with many excellent publications using hippocampal organotypic slice cultures from rat.

It seems that the osmolerity is the problem it may be around 320mosm