Hi Nuri.

My answer falls into a more general scope, but it should be still valid. When patching cells in culture, trypsin could be more of a foe than a friend. Try reducing its concentration or using mechanical dissociation (if possible) to get your cells. Also, people sometimes polish their pipette tip to improve the chance to get the gigaseal. Did you try using this same combination of internal and external solutions with other preparations to see if you can get good seals? That would be a nice control to check out the possibility it's one of them the culprit here.

One last thing: do you have good pressure in your pipette (no leaks) when performing the approach?

Good luck!

I agree with Andre Dagostin

Here are some more suggestion Nuri Acevedo you can review

1: Ensure the recording surface is coated with a cell-adhesive substance like poly-D-lysine or fibronectin, which can help cells maintain morphology closer to in vivo conditions.

2: Passages 3 to 5 are usually acceptable, but fewer passages are preferable, as additional passages can lead to membrane changes that may impact seal formation.

3:Consider using a gentler dissociation method since Trypsin or TrypLE Express can affect membrane integrity.

4: Maintaining cells at 35-37°C during recording and using a physiological buffer with matched osmolarity and ionic composition can also help.

5:After dissociation, allowing a brief recovery period in fresh media can sometimes stabilize the cells and improve seal quality.

6: If these adjustments do not resolve the issue, you might try freshly isolated cells as a comparison to determine whether culture conditions are the primary factor affecting your recordings.

Moreover here is the link for further studies

1: https://yakusaku.jp/pdf/O_253.pdf

2:Article Whole-Cell Patch-Clamp Recordings in Brain Slices