Hello Amir T. Marvian ,

Are you able to give us some photos of how your cells looks?

Did your neurons are clustering to form some sort of aggregates of neurons while they are moving to the edge of the well?

All the best,

Maxime.

Dear Maxime Teixeira,

Thank you for your kind response. Yes, it is right cells form clusters and aggregates, especially at the edges. We use small molecule induced neuroprogenitor cells (smNPC) and then differentiate them by NGN2 overexpression and DAPT, BDNF, GDNF, NT3 treatment. Cells in picture 7 days of differentiation.

I would appreciate your kind recommendation.

Best

Amir

Dear Amir T. Marvian ,

Thanks for your answer and for the photo. From my experience, whenever you see that your neurons tends to regroup and form that clusters, it means that they are stressed and not very healthy.

I experienced that kind of problem and I successfully solved it. Here, I see two factors that may play a role:

1/ Coating. From my experience, 10-50 ug/mL of Poly-L-Ornithine (PLO) should be enough. The critical point is more in the buffer you use, rather than the concentration. For example, if you use dH2O as a dilution buffer for your PLO, it will results in a poor coating because of the very acidic pH you will have. To enhance the coating, I would rather use a Borate Buffer 0.1M pH = 8.5. With this buffer, you will increase the amount of polymers that will adhere to your surface, since the pH will not be as acidic as when it is in sterile water or some PBS.

If you want to use Borate Buffer, remember to thoroughly wash it 4 to 6 times (after the ON incubation) with dH2O before adding your Laminin.

The Laminin concentration you use seems to be enough, but you can try to increase it to 15 to 20 ug/mL if you still face trouble.

The incubation time for both is better overnight.

Also, be sure that you are filling your wells with enough liquid of your coating solution.

2/ Cell number. When the neurons are not enough in a well, they tend to form clusters and aggregates in order to enhance their survival. Since you face trouble, I would recommand to try different number of neurons with your old and the eventually new coating method. Then, you will be able to choose the best option where your neurons are not forming aggregates and moving to the edge of your well.

Hope it helps,

Maxime.

Dear Maxime Teixeira,

Thank you very much for the kind recommendation and insight. I will test it especially the cell density since I am also suspected and I will let you know. Just a short question. Would you think it is possible that using DAPT that prevents glia differentiation would stress the cells? My cells are more than 99% neurons and no glia cells. Do you think the absence of glial cells itself can cause stress?

Many thanks for your support

Best

Amir

Dear Amir T. Marvian ,

In my opinion, the coating buffer of the PLO is the more important point to tackle here, but yes - for sure - the number of cells is definitively playing a role.

I am not experienced with DAPT so I couldn't tell you, but I use Ara-c (Cytosine β-D-arabinofuranoside hydrochloride) at a concentration of 2 uM in my neuronal cutlure, which allows me to have a pure neuronal culture.

It's a source of stress for the cells, but as soon as you have optimized the concentration and the incubation time (2 days for me), you are able to have a pure neuronal culture, without stress clusters. To sum up, I don't think that the absence of glial cells itself can cause stress per se.

All the best,

Maxime.

Dear Maxime Teixeira,

Many thanks for your kind advice which is of great help. Of course, I will also test the coating in the next step.

Best regards

Amir