Hello,

I have sometime experienced the same problem, and most of the time it was because of my coating (Laminine + Poly-D-lysine). Try to make a new one.

Problem of clustering neurons can also for other reasons. For example if you use serum in your culture you should have to try another batch. Growth factor in sera differ from one to another and can affect neurons maturation. 

Cell density in your dishes can also promote clusteringso you can try higher and lower density for your culture.

Contaminations also affect neuronal culture, check your culture media and incubator.

I hope this will be helpfull.

Thomas

I agree with the suggestions above. I always get good primary neuronal cultures when coating with Sigma Poly-D-lysine (P-7280) diluted to 20 ug/ml in 0.15 M Borate buffer (pH 8.5). I leave the PDL solution on the glass for several hours, often overnight, and then rinse extensively with dH2O before plating the neurons. 

 I concur, a good coating is very important.  I would also suggest to trypsinize your tissue (if you are not doing it already) to get a suspension of  well-separated neurons.

Try this coating which is a lot better than PDL.  0.1% Polyethyleneimine in 0.1M Borate buffer pH 8.4.  The solution keeps at 4 C for about 3 months. Heat it to 37C and resuspend any precipitate before using.  Coat 1 hr at room temp and 2 washes of water or PBS.  Let dry.  Works like "super glue".

coating is not good ,  and cell count ,  and the medium change

I agree with Elizabeth that PEI substrate will solve most clumping problems for primary neuron culture.  I used 20ug/mL in water (borate buffer was not necessary) and this worked very well.  You need to wash well with PBS before use since PEI can be a bit toxic at higher concentrations.  In this respect it is interesting to note that embryonic rat neurons do the best, whereas postnatal mouse neurons may not do as well.  However, I routinely grew hippocampal neurons from P1 transgenic mouse pups on PEI substrate with decent success.

As well, I agree with the suggestions above. Coating is very important, I coated the plastic or glass with  Poly-D-lysine in buffer  Borate (pH 8.5). I leave the PDL solution  overnight  and then rinse extensively with PBS (not dH20) before plating the neurons ( at least the last wash is done with PBS). Important I change PDL stock every three months 

thanks so much for reply,,I tried suggestions and I got good attach but still has the problem of neurosphere,, more clumps always,, 

1)which cells number you plate per MWP6::

2) how i will be sure of neuron maturation?

3),, I need pure protein,, I found a protocol use ARA-C to stop the growth of glia,, is it ok

4)which best buffer to collect neuron protein,, and how,, directly using buffer or with scraping firstly

thanks

If your cells are still clumping after 2-3 days either the attachment protocol you use is insufficient or maybe you are plating to many cells.  Use tissue culture treated plates from reputable companies such as Corning or Nunc.   Have you tried my 0.1% PEI suggestion?  I do 1 x 10^6 per well on a 6 well plate.  There seems to be no consensus on when to utilize the neurons for experiment.  Literature varies between 5 to 14 days for experiments.  We do 10 days and beyond but an investigative paper showed that they are not truly mature until 21 DIV.

thanks to reply,, i think attach is ok ,, I used PDL over night 0.1%.. I 'plate 400,000 cell/well in MWP6,, still have very large clumps,, and neuron in the prephery,, I am attaching the photos

Selma, if you started off with a single cell suspension,  then the picture shows that it is the coating that is the problem.  They will migrate towards each other if they are not well attached.  Do you pass your cells suspension through a 40 micron strainer? 0.1 % PDL is a very high concentration as it is equivalent to 1 mg/ml.  Before I started using PEI I was using PDL at 50ug/mL and the highest concentration I have seen in a protocol is 0.3 mg/mL.  Are you using the Sigma PDL?  If yes. it tends to give a non-uniform solution (small granules) that might account for your problem i.e. to much PDL in certain areas.  Try 50 -100 ug/ml of PDL for 1-2 hrs only. 

I agree with all the suggestions above.  Careful trituration is critical to begin with.  If clumping still occurs it's an attachment problem.  I found I had to play with PDL concentrations a lot, and it seemed to vary with different cultures of neural cells.  If all else fails you might try using Primeria flasks from Corning, which work well.  If clumping still occurs it may be  the dissociation technique.  However I don't normally use trypsin on primary neural cells as it tends to be too stringent.

thanks for reply

I used sigma PDL in two concentration

1mg/ml(0.1 %)  and 10ug/ml  >> i got the same clumps,,

what do you think pleease

also

I plated 400.000 c per well in mwp6,, what do you think,,

finally I used 100 micron strainer,, is it too big

thanks

all the best

answers helped me alot

Yes, your strainer is too big.  Go down to 40 micron.

Dear Selma,

clumps are most probably due to pieces of tissues that you kept in suspension. If after trypsinization and trituration you still see floating minces in your suspension just let it stand for one minute before plating. 

Take care that PDL does not dry out overnight leaving exposed areas.

you must plate to many cell, or just look like stem cell

Hello Elizabeth and Jonathan,

Could you please let me know which particular PEI variant (mol. wt. etc. ) you use for the coating ? Info about supplier and catalog number will be very helpful. Thanks.

Sigma-Aldrich cat # P3143 (North America).  Made in Germany as a 50% solution in H2O.  No MW given on the bottle.  I have always coated my plates with a 0.05-0.1% solution in a 0.1M Sodium Borate buffer, pH 8.4.  The coating is at least 1 hour to overnight,  then 2 washes with 2 volumes of H2O and then the plates are thoroughly dried.  Someone suggested that there is no need for the Borate buffer and one can use water.  I tried this and found that the migration of neurons to form small clusters is identical to that obtained with Poly-D-Lysine.  With the PEI in the Borate buffer, the cells give a much nicer monolayer.

Thanks Elizabeth for the quick and detailed reply. I am going to try your method. Is the 50% PEI solution to be diluted to 0.1% in borate buffer for the working solution right before the coating ? Or do you make up 0.1% solution or any higher concentration stock and freeze small aliquots for later use ? 

Is the coating done at room temperature ?

No, the 50% is diluted into the borate buffer in advance & then filter sterilised.  I make 500 ml at a time and keep it at 4C.  When you want to reuse the solution, warm it to 37C,  shake vigorously and place it back at 37C for 30 minutes, shake again.  Repeat this 1 or 2 more time as the PEI aggregates in the bottom and if not well resuspended, your coating will not work.  Give your solution a 3 month expiration date.  I coat in our 37C CO2 incubator just to minimise evaporation.  Hope it goes well!

Hello, how can I check purity and maturity of primary neuron culture?

I need to get pure neuron to check protein expression in neuron cells after some treatment without afraid of astrocytes contamination? also how can I become sure of maturation,, after how long time from starting culture,

thanks

Purity of a primary neuron culture depends of several factors; among them the source of the tissue. Using hippocampal neurons, for instance, employing embryonic brains (E18) largely reduce the number of astrocytes. 

The use of inhibitors of replication such AraC ( at 4 uM) greatly reduce the number of astrocytes.

To be sure of the purity you should employ a W/B against GFAP (astrocities) versus tau (neuronal protein) or even a inmunocytochemistry with the same antibodies

Maturation; basically that depends of the type of neuron, hippocampal neurons mature after two weeks in culture  (If we define maturation as the time that a neurons has multiple synaptic contacts)

Hope this will help

Hi . do you know Poly-D-Lysine solution will destroy at -20 degree or not?

PDL solution is fine at -20 degree for at least half a year. I did it at 10mg/mL. Though I have only tried to freeze and thaw it once only.

Hey Elizabeth, I know it has been a while since this thread was started but have you ever noticed any cell death with PEI?

No, I haven't. My neuron prep is usually at 95-98% viability so there are a few floating cell but the quantity does not increase with DIV. Only 2 possibilies that I can think of, either the PEI is not washed off properly or the water used to prepare the PEI solution or wash the wells is not low or free of endotoxins. This is just deductive reasoning...

I would try Laminin/PDL coating. Additionally, I would trypsinize to break up the neurospheres and replate.

After answering this question 4 years ago, I have acquired new knowledge that I feel is very important to share. First, my 5 year old PEI has started degrading and is actually making the neurons sick so I returned to Poly-D-Lysine. First, the higher the molecular weight of PDL, the better the attachment. Second, the higher the pH, the better the coating. Third, if your coating is lumpy, the PDL is not completely dissoved. So my recipe for neuron "Crazy Glue" is: 70-150K PDL from Sigma (5mg) disolved in 10 ml sterile H2O overnight on a rotary shaker at 50-100 RPM depending on the orbit diameter. Aliquot & freeze at -20C. Coat by diluting 1/10 (50ug/ml) in PBS overnight at 37C. One can use Borate buffer pH 8.4 to coat but my results are fantastic with PBS. My neurons have never been healthier and to date I have survival to DIV 24.

Hey Elizabeth. Thanks for sharing your method. I do have a question, though. You said that your 5 year old PEI has started degrading and making neurons sick so you returned to PDL. What was the reason that you switched to PDL instead of buying new PEI ?

And using only PDL alone was sufficient to make neurons without forming clumps? If so, what was the longest period that you cultured neurons?

I originally switched from PDL to PEI for cost purposes. I was just starting to make primary neuron cultures and they were just as healthy on PEI versus the badly dissolved 30-70k PDL. My decision to not buy another bottle of PEI was 3 fold. First, there was no guarantee that another lot number of PEI would perform identically to my first lot and the product is not cell culture tested. Second, the neurons adhere just as well if not better, are much healthier and live longer on the completely dissolved 70-150K or 300K PDL while coating in PBS. Also, Soneela Ankam (above) appears to have had the same problem with PEI as the one I recently encountered. Finally, why would the PEI degrade? It was much better to remove that variable from the primary neuron preparation.

I do get some small gathering of neurons i.e. about 25-50 neurons migrating towards each other which I can only see after 10DIV so this is a slow process. This seems to occur when I use frozen aliquots of PDL versus freshly dissolved. The freezing might fragment the long strands of PDL (just a hypothesis). If you feel that this is a hindrance in your assays then try a well dissolved 300K PDL coated in 100mM Borate buffer pH 8.4. Another worker whom I consider the queen of TC has tried my improved PDL coating successfully with primary muscular neurons that would only attach to Matrigel.

As I mentioned before, I got my neurons to DIV 24 without much effort. Please note that the older they get, the more often you have to change the media as it acidifies quickly. I am certain that with a little care, they would have gone longer. I still have to tackle the problem of the drop in % moisture after the incubator door is opened. This affects the osmolarity of the media and the neurons are sensitive to this type of schift.

Thanks for the reply back, Elizabeth. I appreciate your help a lot.

So based on your experience, PDL also does good to neurons or even better since you observed toxicity of PEI to neurons.

Some additional questions if you don't mind,

1) Do you use PDL only or with any other coating substrates like fibronectin, laminin, or matrigel?

2) So are you saying you don't observe cell gathering when PDL was prepared fresh?

3) You emphasized that complete dissolving of PDL stock is important and told us to dissolve it in sterile H2O overnight on a rotary shaker at 50-100 RPM. What container and temperature do you use when dissolving?

- Byung

Hi Byung,

When the PDL is prepared and coated as I mentioned in another answer, the neurons do much better on the PDL then on PEI.

1) I do not add any other coating with the PDL.

2) There is less gathering when the PDL is fresh and even less when I used the 300K PDL, but neurons just love each other....

3) I use the Sigma PDL, US cat #P6407- 5mg which is in a sealed bottle and under vacuum. In the TC hood, using 70% EtOH, I sterilise the metal lid and then peel it back to reveal the rubber septum which I also sterilise. I inject 10 ml of sterile Type I water through the septum with a syringe and needle. Then I place the bottle on the rotary shaker at room temperature overnight. The dissolved PDL is aliquoted into 1ml and frozen at -20C. I dilute the PDL 1/10 in tissue culture grade PBS to give 50 ug/ml. I coat for minimum 3 hours but mostly overnight in the CO2 incubator to prevent evaporation.

Hope this helps.

Elizabeth

Elizabeth,

Yes that helps a lot.

FYI, My concern is that neurons aggregate so well over time so it is sometimes hard to study individual cells with IF or live cell imaging. Some say that neurons do cluster because that is just the way they survive better but others bring up coating issues, mainly saying that coating is not strong enough or degrade over time so can hold neurons tight and the cells tend to get close to each other to survive.

Thank you for the method. Will try that. Hope this makes neurons attach better.

Byung

I usually plate at high density for assays but I recently seeded some 35mm imaging plates at 250,000 neurons/dish. At least 50% of the neurons were individual and I could have seeded higher.

Elizabeth

How many neurons do you usually seed? And for the 250K seeded dishes, I guess you have cultured them for short period of time and they will likely to aggregate more and more over time?

Hi Elizabeth, thank you for all your comments, this is a very helpful thread.

What do you think the maximum concentration of a PDL solution should be to get a completely dissolved and high quality stock? The protocol I've been using makes 5mg/ml PDL in sterile H20 and I occasionally will see undissolved PDL when coating. That may be rectified by moving to a higher pH solvent like the Borate buffer but I'd like to hear your thoughts as well.

Hi,

I culture E18 rat cortical neurons. My lab was having similar adherence problems with our cultures when coating with PDL (10 ug/ml in ddH2O, coated at 4C overnight). Some of the cultures would spread out on the plate and be fine, but most of them would form these huge clusters of cell bodies in some spots and have bundles of processes floating in the media above the plate between these bundles. The cultures that were good would deteriorate after ~2 weeks. We tried coating with higher concentrations of PDL, but the same thing happened.

Then we switched over to dPGA from Dendrotek (https://dendrotek.ca/products/centrifuge-tube) for coating our plates and it solved our issues. Way more consistent adherence, cultures lasted longer and seemed to have more processes on the plate. We use it the same as PDL, 10 ug/ml in ddH2O at 4C overnight. I've gotten cultures to last for 10-11 weeks using this.

Hope this helps!

Thank you Teddy.

It was a long time ago, but it might help someone else. Maybe us, as we will start other experiments on neurons this year!