Good day, Seyedeh !

I work with primary cells, including neurons.

Neurons are very capricious cells. Rather, their death is due to the very process of reseeding, because when you rip the neurons off the substrate with trypsin, the expression of membrane adhesion molecules is disrupted. And on day 5, your cells are already differentiated, that is, with processes (mature), in this state the cell has formed a phenotype of molecular markers, including molecules of intercellular and cell-bonded adhesion, when these molecules are damaged, it is very difficult for a neuron to restore them (it is easier to recover immature neurons).

The second reason is the number of cells - for the survival of neurons, a sufficiently large number of cells is needed so that the cells can form intercellular contacts through the mechanism of paracrine regulation. The destruction of these contacts during reseeding also leads to cell death, since neurons form a neural network with each other, which, among other things, provides their trophism (nutrition).

Alexandr,

I appreciate your well-described answer, I will use 1x trypsin again to see how it goes... I seeded 10,000 cells/well in 96-well plate, do you think it's ok? however, I guess, considering a large number of dead cells, a high percentage of the cells I counted as living cells may have died after plating and the mature ones are less than I expected!

Alexannder, what do you think about a problem in coating plate? could a weak coating lead to the cell death like this after 7 days?

thanks in advance

may be the surface of you 96 wells is not sufficient to allow the extension of neurites, usually we carried out primary cultures of mouse neurons on either 6 or 12 wells plates coated with laminin and we keep these cells for 10-12 days before they detached and die.

thank you Dominique for your response, because I'm going to conduct MTT assay in different treatment groups, I have to use 96-well plate for culture and because there are also other articles that have the same method as mine, this point you mentioned didn't come to my mind, however, I will definitely consider it if I face this problem again even with a trypsin change and increasing PLL exposure time.

I would suggest longer time if coating. But also removing glutamine after cells settled properly (like 3 days in vitro). Immature neurons are not sensitive, but as the cells get matured the glutamine get cytotoxic. Good luck!

thank you Oksana,

It's a very good suggestion, I'll try it👍

Dear Farzaneh

There are two kinds of 96-well plate that I have worked with them, round and flat bottom one. You can try both them. I am not sure, but you can check it. Good luck.

Thank you dear Maryam

As far as I know, because these cells are adherent, I should only use flat bottom ones, shouldn't I?

Dear Seyedeh,

it might be a problem of the number of cells you seeded in the beginning as mentioned earlier. Since the cells attach properly, I do not think that the coating is the problem (although ofc they can always be problems of neurite extensions when the coating isn't done properly). However, mostly when the coating is not done properly, neurons do not attach at all. Thats not your case.

Neurons need a certain number of cells surround them and their direct cell-to-cell contacts. If they are missing, they will die. The initial seeding concentration is crucial for neuron survival. For me it looks like you should seed some more cells (try with the double amount). Since neurons get more fragile the more mature they become some neurons may die during differentiation which lowers neuronal numbers and leads to cell death.

Good luck with your experiments!

Lena

Dear Lena,

I appreciate your valuable response,

I have been testing the previously mentioned factors but it's on first days yet and too soon to judge the result, however, I'll focus on the number of the cells, if my efforts don't work this time.

thanks😊

Dear Seyedeh,

A media change is a very sensitive time for primary neurons. You may want to read this RGdiscussion:

https://www.researchgate.net/post/Why_do_my_primary_neurons_disintegrate_in_my_cultures

As Lena and Alexandr mentioned, it can be difficult to get the correct plating density. Too few and they die unless you add trophic factors, and with too many they can be very susceptible to excitotoxicity. I always calculate the number of Trypan-blue excluding (live) cells for my preps, since dissociation conditions may vary, even with the same pair of hands doing the dissociations.

However, I have always dissociated my tissue either with Papain (inhibited with Ovomucoid afterward), or simply with calcium-magnesium free PBS (depending on embryonic age and neuronal type). In order to use Trypan blue successfully you must be gentle with the dissociation so as not to make the cell membrane too leaky. Otherwise you get a false sense of the number of dead cells.

Unfortunately, the smallest wells I have ever used is a 48 well plate, so I am not able to suggest a good plating density.

To get a good density for the 48 well size I scaled down from my 24 well plate density based on the area, not the volume, of the wells. I then tried a few different densities based on that number to see what worked well.

Good Luck!

Jill

Dear Jill,

thank you for your helpful advice,

It was weird that my cells started to die only several hours after media exchange (1:1) on day 7, I'm not sure if this problem relates to the cell density, but I'll try different cell numbers, if this problem doesn't solve.

Dear Seyedah,

Delayed neuronal cell death is one of the hallmarks of excitotoxicity, since a takes a while for calcium ion entry and for the energy gradients to run down as the cell tries to buffer the effects. You might want to look at the papers from Dennis Choi's group in the 1990's. They set up an excitotoxicity assay system using embryonic mouse mixed cortical cultures (includes glia) to screen for compounds that might work in the treatment of stroke. They showed the difference between excitotoxic cell death based on glutamate and calcium entry and the more rapid cell death due to sodium entry. They also adopted their system to 96 well plates, but they had a robot that they used for those assays. The group in which I worked tried to adopt their assay to 48 well plates in order to test more compounds, but ended up returning to the 24 well plate format. Since we were quantifying cell death (or survival, depending on how you think about it) with LDH release, we were able to remove an aliquot of media to the 96 well assay plates, rather than plate into 96 well plates.

Usually in those mixed cultures the neurons are not vulnerable to excitotoxicity (tested with either glutamate or NMDA challenge) until the neurons mature somewhere between days 10-14 in vitro. It is possible that your neurons have matured more quickly, since I have no idea of the embryonic age or type of neuron you are trying to culture. That is also why I pointed to the RG discussion that suggested lower levels of glutamate for maintenance.

Again as regards the age & type of neuron, the point at which they become dependent on trophic factors may also be a factor ( I am only familiar with the work on work on purified retinal ganglion cells by Anke-Meyer Franke & Ben Barres). However, as mentioned by by others above, density plays a role in cell survival by production of necessary factors & activity. That is probably one reason why some researchers have found that they have better neuronal survival by delaying media changes.

I also forgot to mention that your coating protocol is probably fine, since you still have cells and processess attached on day 8. I prefer to use poly-D-lysine since it is the form that cannot be metabolized, but there are plenty of researchers that use PLL.

Good Luck! It can take some time to work out the successful culture conditions for your type of neuron & plate format, so don't become discouraged.

Jill

Dear Jill,

I'm so thankful for your good explanations and introduced references,

I have forgotten to mention that I'm working on primary neurons isolated from one-day rat neonates, I think you're right, based on what you and Oksana said these cells are probably more susceptible to excitotoxicity, and I think it's better to eliminate glutamine from the feeding media formulation earlier on day 4, when I want to add new media.

I hope I can set up this method in our lab with the help of your valuable experience,

Thank you so much🌼

Dear Seyedeh,

It may not be possible to eliminate glutamine totally, depending on neuronal type.

As you will find, there is a great deal of literature on neuronal cell culture. There are numerous threads here on RG.

I also suggest that you look at the work of Gary Brewer on hippocampal neurons for some helpful insights on dissociation techniques and media formulations, as well as their use in a variety of assays.

Although it is older, it is well worth obtaining a copy of the Neuronal Cell Culture book edited by Banker and Goslin,

and reading the different chapters on neuronal culture--see if your institution has it in the library, or can get it for you by intralibrary loan. It also used to be available on line. In it you will find the chapter by Huettner and Baughman on their techniques for dissociating and culturing postnatal cortical cells from visual cortex. It was also used as the basis of the Barres protocol for postnatal retinal ganglion cells, and uses papain rather than trypsin.

While the Huettner and Baughman protocol is similar to the protocol on the Worthington (source of the papain) and Life Technologies (ThermoFisher) website, it has a dissociation medium that contains Na2SO4 and K2SO4, which are supposed to help keep the neuronal ionic gradients intact. It also uses a lot of different blockers of glutamate excitotoxicity during dissociation, which can be critical for neuronal survival. I found both of these to be very helpful in increasing yield of live postnatal neurons for plating and culture. Depending on what question you are trying to address, it might be useful to have a culture system that contains representatives of all neuronal types from the brain area of interest.

If you are starting with PN0 or PN1 rat brain (area??), it is certainly possible that by DIV 7 you are looking at neurons that have matured to the point of both excitotoxicity susceptibility as well as dependence on target derived trophic factors. If you are working on postnatal cerebellar granule cells, there is a huge body of literature on their culture. However, I have not isolated those particular neurons and so I am not able to offer any advice. I think Vance Lemmon's group has a system where they culture them in a 96 well format.

Good Luck and happy reading.

Jill

Dear Jill,

your tips are very helpful,

I intend to work on the mechanisms of a toxicity on primary neurons and for now it doesn't matter what specific origin of the brains is selected so we decided to use the whole cortex for the first steps, up to now I've been using similar articles, experiments online and even some videos to obtain related information, however as you now these sources aren't very accurate in the details of their methods and now I feel, as you suggested, I need to take a more in-depth look at the book references to learn the basic principles of this method,

Unfortunately, I don't have any access to these kinds of books entitled 'primary neuronal cell culture' and I can't even buy them online from foreign websites (I don't know how aware you are of the Iran's limitations),

so can I ask you if you could send me this book you mentioned, if you already have its pdf format or if there is NOT any problem for you?

thank you in advance

have a good time

Dear Seyedeh,

Unfortunately I do not have these publications as PDFs-I only have them as paper reprints.

One of the reasons I suggested these references is that not only are they IMHO classics, but I was hoping they would be available to you since they are older and were published in journals in print format that may be on the shelves of your Institution's library. Or, if you don't have library access, in the files of some of your Neuroscience faculty.

Another source to which you might have access is Current Protocols in Neuroscience from Wiley (publishers). While it is now online, it used to be available in a paper format to which libraries subscribed. It has several chapters on culture techniques.

One good thing is that there is a wealth of information here on RG on cortical neuron culture, and there are several protocols that have been uploaded. I think there is even a link to a JoVE video in this thread:

https://www.researchgate.net/post/What_is_the_best_way_to_enzymatically_dissociate_mouse_brain_tissue_that_gives_a_high_yield_for_all_cell_types_neurons_astrocytes_microglia/1

As for specific details of methods, as you found you can certainly ask those questions to the RG community. For instance, if you are still having problems with a lot of debris in you dissociations, you can look at the thread on the use of a BSA cushion

during centrifugation: https://www.researchgate.net/post/Why_is_my_primary_mouse_neuron_cell_yield_so_low_when_doing_a_BSA_cushion_purification_step

Good Luck with your experiments--

Jill

Dear Jill,

Many thanks for your guidance on finding references,

I'm working at faculty of pharmacy, so in our library, pharmacy professional books are mostly available, and the specialized books in the field of neuroscience are not easy for me to find, I'll work on the other ways...

anyway, Thank you for taking the trouble to help me.I do appreciate it😊

‌I wish you luck dear friend 🌼

Dear Ghulam,

Thank you for your answer