Thank you all for your answers.

- Daniel, we don't use organotypic cultures for electrophysiology only, we also use them to evaluate cell viability after Oxygen and Glucose Deprivation and we also perform immunofluorescence, protein extraction and RNA extraction on them. We do not destroy the insert, so we thought that we might be able to reuse it. We were given a protocol, but in our experience, recycling the inserts is not convenient because liquid gets through the pores and floods the top of the insert.

- Ulrich, there is another method to fabricate your own inserts in Koyama R et al., 2007, J Pharmacol Sci.

For anyone interested, the protocol for recycling the membranes is the following:

1. Remove the cultures from the insert gently with a spatula or brush

2. Rinse inserts in tap water

3. Place inserts in Trypsin-EDTA (0.05% trypsin, 0.02% EDTA in 0.9% NaCl or HBSS) (we use leftovers from cell passages)

4. Incubate 1h 37º in incubator.

5. Rinse 3 times in demineralized water

6. Place in 70% alcohol for at least 24h

7. Dry alcohol under the hood and store under sterile conditions

I don't have an answer to your question, but I'm curious as to what you use the inserts for that you would even be able to reuse them. In our application, the insert is destroyed when we cut the organotypic slice out to put it in the recording chamber.

Hi Federico and Daniel, I have not found a way to do this, but check out this paper - it works:

Stoppini L, Buchs PA, Muller D (1991) A simple method for organotypic cultures of nervous tissue. J Neurosci Methods 37:173-182

We used it for organotypic cultures of cerebellar slices that were then recorded.

Sorry, that was the wrong reference,it is of course the original Stoppini paper using millipore inserts. Luc had another approach using 'confetti' cut from bulk membrane placed on culture medium with a bit of agar (1%). I'll look for the original paper describing it. Maybe I can also dig out the methods section of a diploma thesis, in which we used this successfully.

No its only for one time use. Can't be cleaned and sterilize .

Thank you all for your answers.

- Daniel, we don't use organotypic cultures for electrophysiology only, we also use them to evaluate cell viability after Oxygen and Glucose Deprivation and we also perform immunofluorescence, protein extraction and RNA extraction on them. We do not destroy the insert, so we thought that we might be able to reuse it. We were given a protocol, but in our experience, recycling the inserts is not convenient because liquid gets through the pores and floods the top of the insert.

- Ulrich, there is another method to fabricate your own inserts in Koyama R et al., 2007, J Pharmacol Sci.

For anyone interested, the protocol for recycling the membranes is the following:

1. Remove the cultures from the insert gently with a spatula or brush

2. Rinse inserts in tap water

3. Place inserts in Trypsin-EDTA (0.05% trypsin, 0.02% EDTA in 0.9% NaCl or HBSS) (we use leftovers from cell passages)

4. Incubate 1h 37º in incubator.

5. Rinse 3 times in demineralized water

6. Place in 70% alcohol for at least 24h

7. Dry alcohol under the hood and store under sterile conditions

Hi Federico! Could you please tell me where you found this recycling protocol? Is there a reference?

Thank you everyone!

For those that recycle the inserts, do you find that using the biopore membranes affects cell health or transfection via biolistic methods?

Hi, I was working with 300 um thick organotypic slice cultures on PICMORG50 inserts. I actually had problems with the formaldehyde fixation as the slices detached from the membrane, became extremely fragile and were lost during subsequent dehydration and clearing steps. Since then I have only done snap freezing to preserve anatomic landmarks. You guys here probably managed to overcome this problem, so I am really curious how you did it?

Hi Bertalan,

 I have experienced that issue with the membranes occasionally.  I found that reducing the speed of the shaker prevented the slices from detaching.  Fragility has not been a significant problem for us.  Perhaps you can increase the thickness of the slices slightly so they will be less fragile?  Good luck!

Amber

Hi Amber,

thanks for your quick reply. Yes, I went through the same troubleshooting, PFA fixation was no problem with 300 um thick slices without shaking - I carefully pipetted the fixative on top of the slice and below the membrane - but in the end there were no samples left to visualize a nice coronal section from cortex to basal ganglia. I could try increasing OBC thickness, which, however reduces their viability (Mewes et al. Organotypic Brain Slice Cultures of Adult Transgenic P301S Mice—A Model for Tauopathy Studies). Do you slice the brains or do you use confocal?

Cheers,

Bertie

Hi all. I hadn't seen this question in a while. To asnwer some of the comments:

1- I got this protocol from a colleague in Italy. He said that this is the way they've always done in the lab. I guess there's no mention to recycliyng membranes in papers.

2- To fix, I used to put the inserts directly in PFA for 1 h plus a small drop of PFA onto the slice. In this case I would not reuse the insert. Then I would carefully detach the slice with a brush and PBS, and subsequently wash the, and perform immunohistochemistry as free floating sections. Imaging would be done in confocal, since the slice is quite thick (we cut at 350 um, and it ended in 150 aprox.)