I've been culturing neurons for 20 years and can tell you, there are lots factors involved in healthy neuronal cultures.   However In my experience, I only see aggregates/spheroids when the polylysine has gone bad.  We use a similar concentration and coating protocol, so can't help you there.  However we make a batch in a borate buffer pH 8.5 and freeze it away at -20C (one time use).  Another difference is we plate neurons for 1-2 hours using DMEM + 10% FBS before switching to Neurobasal + B27 for the rest of the culture.  This improves cells viability quite a bit and we routinely grow neurons for 4-8 weeks.  Glia are always a problem- many researchers try to kill them off using something like AraC. 

Hi Marcela,

sometimes it is happen to my culture, and the cause are too much, but the principal causes are the coating and also B27 supplement, what i can do for you is send to you my protocol.

About the glial cells I tried to kill by AraC, but it doesn't work properly in the sense that also the neurons are not in good condition because they need for survival of the factor produced from glial cells.

Marzia

thank you a lot guys, for your fast response.

Yes, Marzia, if you could provide your protocol, that could also help me, please. Then I can compare the differences. 

Bryen, did you mean regarding the borate buffer, that you dissolve poly-lysine powder in that buffer and let it freeze? I am dissolving it in water, making 10X stock and then preparing 1X again with water. 

I think the problem is really with coating, since I hear the same from other people, but I need to consider other factors as well. It is strange since the cells look pretty nice after 2-3 days and then they screw up somehow, that makes me think about something else than just the coating. 

I fully agree with Bryen on the diagnostic and solution. The cells look nice initially because there is enough PolyL adsoption to the plastic but the film is too weak and breaks down within a few days, causing the neuros to stick to each other and forming balls. The common cure is a borate buffer and overnight incubation at 37. You may also make sure that your plasticware is "for culture" (ie the surface is modified to charge it and make it hydrophilic. untreated polystyrene preventsprotein binding). I hope this helps.    

Hi Marcela, I have been preparing primary cultures from both cortex and hippocampus for several years now. I can tell you that much, what works for me using 24 well plates does not translate at all into 96 well plates. You have to find the right balance between cell number and volume of media. The later is a real bottleneck when it comes to culturing in 96 well plates.

thank you guys. It seems that 96 well plates and coating and culturing neurons is a real pain. All plastic ware I am using is for culture, however, 24 well plate looks nices, but also depends, some wells look ugly while the others much better, but generally compared to 96 well plate they are uncomparable. Yes, Oliver, I completely agree, there is something with 96 well plate which makes it difficult.

Hi Marcela, I agree that the sun-like aggregates are a sign of poor attachment to the surface. I would recommend that you coat with mouse laminin after washing off the excess poly-D-lysine. Also, have you tried plates from another manufacturer? There are subtle differences in adhesiveness.

You don't say what medium you are using; there are alternatives that may be better for long-term survival of primary neurons.

Hi Richard,

I am using Neurobasal medium + B27 supplement + GlutaMax. 

Hi Marcela,

I have used Neurobasal/B27 and my primary cultures are usually good for at least two weeks in it. But if you want healthy neurons for three weeks or more (and faster neurite outgrowth), please try NeuralQ/GS21 (see link).  Sorry for the commercial, but I consistently see healthier neurons in this medium that my company has developed.

http://www.globalstem.com/neural-cells/neural-cells-media

Hi Marcela,

The small round cells are likely dead neurons that did not survive the preparation.  They are not likely microglia if you are seeing that only 1 day after plating.  It could also signal that your coating method is not good.  If cells clump together after several days and the neurites form bundles, this is a clear sign that the coating is not adequate.  With E18 rat embryos you should be able to get much better survival and dispersion of the cells on the substrate.  The cells should stay alive and healthy for 3+ weeks.  You can check the osmolarity and pH of your media, the quality of your batch of FBS and B27 or similar supplements, as these have a big impact on the results.  However, I think your main problem is the coating substrate.  I would highly recommend using PEI Max 40KDa from Polysciences as the coating substrate.  I see a huge improvement when I use this instead of PDL.  I make it up to 20 ug/mL in pure water, pH adjusted to 7 and sterile filtered.  Too high of a concentration can be toxic to neurons, similar to PDL or other substrates.  This works best with embryonic rat neuron cultures and may be a little harder with same age of mouse neuron cultures. 

thank you a lot for sharing your experience, Johnathan, I will try to modify few things and see whether I can get any improvements.

The PEI is very cheap in comparison to the PDL, so it will save you money in addition to being a better substrate.  Try washing with 1X PBS  or DPBS instead of water, regardless of the substrate used for coating. 

The density you use for 96 well plates is the same as I used for 24 well plates.  I think you can reduce that plating density substantially once the survival issue is improved. I think the protocol you use looks sound, but there are some things that are difficult to describe such as how well to triturate the tissue and how to properly prepare the glass fire polished pasteur pipettes for the trituration steps. 

trivial question Jonathan, what exactly is PEI? Well, for triturating, I am using regulat 1 ml pippete tips, no glass. i am using high density plating since for calcium oscillation measurement you have to have cca 50,000 cells per 96 well, to be able to see baseline oscillations and then treat the culture with some drug. 

Hello Marcella,

I have cultured both mouse and rat embryonic cortical and hippocampal neurons for many years.  In regards to the large number of unhealthy cells that you find at 24 hrs in vitro, I noticed that you mention that you are using 1 ml pipette tips for triturating your tissue. This can cause a lot of damage, which translates to neuronal death.  While the polypropylene tips are more slick than glass, you are limited in the size of the opening.  In addition, not all tips have a nice edge, but can be a bit ragged.  Also, unless you are using a motorized pipetter at a very slow speed, it can be difficult to triturate the material slowly by hand, and not get bubbles in the mixture.

I agree with Jonathan, and suggest that you switch to Pasteur pipettes for trituration.   Technique is very important, and you need to gently and slowly dissociate the material.  I make three different sizes of opening, and I use pipettes made of soda-lime glass rather than borosilicate glass so that it is easier to control the size of the opening. (I used to siliconize the pipettes, but now I just make sure to pull some of the buffer with BSA into them first in order to keep the cells from sticking to the glass).

I was also trained not to keep triturating already dissociated cells.  So, we would triturate very gently with the pipette with the largest opening until it looked like the dissociation media was a bit more cloudy, then let the large pieces settle out (maybe 30 seconds to one minute?).  We would then remove the supernatant (with the dissociated cells) and collect it in another 50 ml centrifuge tube.  We would then add more dissociation media, gently triturate using the second pipette with the next smaller opening, add that supernatant to the collection tube, and so on.  Any material that was left after the third trituration we did not use, but discarded, since in our experience those neurons would be dead.  I don't recall our cell counts off hand, but we had plenty of neurons from a litter of E18 pups. 

Good luck!

Jill 

thank you for your advice, Jill.

Hello Marcela,

I came across your thread while looking for answers re: what appear to be interconnected spheres like you subscribe. Bryan mentioned coating deterioration as a likely culprit and I think that is the case for me. Yesterday I called MatTek regarding two cases of Poly-d-lysine glass bottom dishes and it turns out they were made in 2015. MatTek only guarantees them for one year. So, I've just been lucky until the last few weeks. I'm trying to put together a protocol for acid washing 15mm circles, coating them with Poly-d-lysine for 12 well plates. Any and all suggestions welcome.

Hello, i usually do the cell culture and i would like to share my protocol also

Why do we isolate the neuron of embryonic rat not adult??

Adult brain is difficult to dissociate without killing neurons, as there are many more processes. Fetal neurons tend to survive and differentiate more readily, and are more appropriate if you are interested in development. There are protocols for culturing adult neurons, but I've never tried them.

Look into Marani E et al., 1988 Ionic conductances in cultured pre-infundubular cells from the hypothalamic arcuate region. Neuroendocrinology 48: 445-452 for methods. Postnatal rats till p6 is also possible for cortex. For behaviour of these cultivated neurons see also T.Heida, 2001, Dieelectrophoretic trapping of dissociated fetal cortical rat neurons. IEEE Trans Biomed Eng 48: 921-930.