Primary neuronal cell culture: Key ingredients to tricky techniques

Amy Palubinsky

Neuroscience has come a long way since Cajal perfected Golgi’s staining methods, gracing us with those early sketches of individual neurons in the late 1800s. However, for a field still considered by many to be in its infancy, we’ve certainly got our work cut out for us—particularly if your research involves primary neuronal cell culture techniques. Over the years, there have been countless chapters, entire books, white papers, and journal articles dedicated to primary neuronal cell culture: what to do, what not to do, and, of course, how to do it. Unfortunately, there is no standard mold. Every neuronal cell type has its own particular requirements—components it prefers over others. Heck, even the same cell type is cultured differently across labs. So where do you start? What are the key ingredients?

The culture of primary neuronal cells is particularly challenging since most mature neurons do not undergo cell division. This means that you must start with freshly dissected tissue dissociated into a cell suspension, select for your cell type of interest, and maintain those cells in culture until you’re ready to experiment on them—after which they’re gone and you get to start all over again (and again and again and again).

To circumvent this, we can sometimes use immortalized cell lines derived from neural tissues. These lines are advantageous, as they can be grown fairly easily and amount to pretty much an unlimited supply of cells. Some of them can even be differentiated into a more mature, post-mitotic cell. However, differentiated or not, cell lines often present with different phenotypes, responses to treatment, etc. than the cell type from which they were derived. This is one of the major reasons why the use of primary cultures is often more desirable—because they are more likely to recapitulate the same properties they had in vivo.

So, you still want/need to culture primary neurons? Here are some important things to keep in mind:

Starting material
What area/cell type are you trying to grow? The age of the animals from which you take your starting tissue is key. Typically, it’s easier to culture from embryonic tissue because its cells are still developing and tend to have more plasticity/resilience than a more structured post-natal tissue. But the exact embryonic day can also be important. For example, you can’t culture specific hippocampal neurons from embryonic tissue that has not yet even developed that particular region of the hippocampus.

Dissection, digestion, and dissociation
Every lab does it differently—and the end result is a neuronal cell culture—but here are some factors to consider for these steps. Do you dissect on ice, at room temperature, or at 37°C? What enzyme do you use (as some enzymes are better suited for embryonic versus post-natal tissue digestion)? After you’ve chosen your enzyme based on its optimal efficiency, do you digest on ice, at room temperature, or at 37°C? Now that you’ve chosen the temperature for digestion, how long to you digest for?

So now your tissue is digested. Sweet. Time to dissociate into a cell suspension. Protocols will say something like, “triturate the tissue using a pipette.” OK. A glass Pasteur pipette? Does it need to be rounded at the edges (i.e. fire-blown)? A plastic pipette? 2mL, 5mL, or 10mL? The diameter of those holes is certainly very different, and it’s not like you can see the cells. So up and down five times? 10 times? 100 times? Yikes, right?! But are you getting the picture?

Culture vessels and substrates
Imagine you’ve dissected out an area of the brain you’re interested in and thrown it into some enzyme to digest, and then tediously dissociated it to have single cells in suspension. Now you want those cells to happily grow on a glass or plastic dish. Really? Caveat one: Some neurons prefer glass over plastic, some favor plastic over glass, and the options don’t stop there when it comes to plastic. Which type? Caveat two: Cells that were once part of an intricate scaffolding system are going to need something to stick to—something to grab onto while they attempt to re-establish neuronal processes and send out their dendrites to meet the neighbors.

Another, almost limitless set of options: Poly-D-lysine, Poly-L-lysine, collagen, fibronectin, laminin or, if you’re really lucky, some combination of them. Not to mention what these substrates are reconstituted in. Water? Sure, for some. But others may require phosphate buffered saline, borate buffer, or cell culture media. Some need to coat the culture vessel for days; others for minutes. But in the end, the choice of substrate can be the difference between 10 percent of your cells sitting down and establishing connections or 95 percent doing so.

Plating density
Some cells need neighbors in close proximity to make a happy culture. This is great, unless you want to do some immunocytochemistry and you have so many dendrites that your images look like hair, and you can’t make sense of anything. Others cells would rather have some distance and just extend a dendrite to talk to a neighbor when they feel like it. This is also great, unless they have so few neighbors that as soon as they become unhappy (i.e. you treat them with a chemical) they fizzle out and die well before they should (based on that other lab’s paper).  

Media components
A delightful topic, and one that is constantly debated: Dulbecco’s Modified Eagle Medium (DMEM). OK, which one, because there are seemingly billions to choose from. Neurobasal? Again, which one? How much glucose do you want in there? 25mM seems a bit high for a cell that came out of a brain where it sees way less than that? Do you need to add glutamine? Glutamax? What the heck is the difference, anyway?

Check out the debate over the decay of glutamine in culture media. Do you add it fresh, or just assume that what the company says is in there is still there by the time you open the bottle? Do you use serum derived from an animal or a serum supplement concocted by a company (sometimes with sub-standard quality control)? Which company? What do you want in your supplement? Antioxidants? Which ones? Insulin? How much of each? Do you use the cookie-cutter versions or make your own at precisely the concentrations of everything you want/need? And this is before we even get to growth factors!

Experiment time
How long do you have to grow these things before they express the appropriate receptors/proteins/RNA/transcription factors you need to see activation of the pathway you’re trying to interrogate? Is there something in your media confounding your results? Are your cells plated too densely? Too sparsely?

What a cluster this all is, right? And depending on your culture technique and experimental parameters, these all constitute areas where reproducibility can vary greatly from culture to culture, even in the same person’s hands—let alone between labs. My advice? Seek out someone whose work you trust, learn from their lore, write insanely detailed protocols, and recognize and accept that sometimes it really is just voodoo.

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Amy M Palubinsky

Amy Palubinsky is a Neuroscience PhD candidate at Vanderbilt University and mom to two human children and one fur-kid. She hates mornings and therefore loves coffee.

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