Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Between arduous isolation procedures and finicky culture conditions, neurons have the reputation of being some of the most challenging cells to grow. This reputation is not entirely undeserved. Yet the availability of frozen cells and off-the-shelf reagents has enabled non-experts to explore the burgeoning field of neuronal cell culture.
From whence?
There are three principle sources of neurons for research, says Navjot Kaur, staff scientist in cell biology at Thermo Fisher Scientific. Historically, neurons were freshly isolated from rodent brains or spinal cords. More recently, vendors have made frozen neurons available for purchase, and researchers can also derive neuron-like cells from pluripotent stem cells.
Two intertwined considerations to take into account are the cells’ origins and the types of experiments for which they will be used. For biochemical studies that require large numbers of cells, it might be attractive to look at neurons from the cortex—the largest part of the rodent brain. “But if you’re looking at more stereotyped growth behaviors, or you want a restricted population, then you have to start thinking about how to isolate a specific region that might have more select cell types ... that are going to respond to growth factors and cues, in a more homogenous fashion,” says John Henley, associate professor of neurosurgery at Mayo Clinic.
Some researchers use retinal ganglion cells, for example, Henley says, though his lab typically focuses on the hippocampus which is important for learning and memory. They start by dissecting out the hippocampus—“an easy-to-find structure in the brain”—and then dissociate the neurons and grow them in culture.
The tried-and-true procedure to dissociate neural tissue is to mash it through various sizes of mesh screen, which inevitably leads to “a lot of cell death and debris,” Henley says. An easier and more common method is to heat the tissue in the presence of an enzyme solution; several vendors offer kits for this, including Pierce. Enzymes may wreak their own collateral damage, though, cleaving off surface receptors that can be important for adhesion or recognition of growth factors, for example. “So it becomes more important, then, to plate those cells in culture and grow them for a period of time … before you start doing experiments on them.”
Rodent neurons typically are harvested at the late embryonic (or perhaps early post-natal) stage—by which point their fate has largely been determined. “The further along in development, the more consolidated the brain gets; as the neurons develop, they put out more branches and start to integrate with each other,” making them increasingly more difficult to dissociate without damaging the cells, explains Sam Lloyd-Burton, product manager for STEMCELL Technologies’ neuroscience products. “By using neurons from older animals, you’re not speeding anything up, you’re just going to get lower viability, because you still need to go through the process of allowing the neurons to mature in culture.”
Brrrr!
Researchers can purchase pre-dissociated neurons either fresh (refrigerated) or cryopreserved, and shipped overnight, from several sources (eg here and here). Most common are cortical or hippocampal neurons, but vendors such as Lonza also offer cryopreserved cells “guaranteed to have been isolated from certain brain regions,” such as the cerebellum, striatum, hippocampus and hypothalamus, as well as the dorsal root ganglion, notes Lubna Hussain, Lonza’s senior product manager for animal neurons.
“We QC them—we have a battery of criteria that we meet to show that they are cortical or hippocampal cells or striatal cells,” and that they are equivalent to freshly isolated neurons, Hussain says. In addition, cryopreserved, commercially sourced neurons enable experimentation on consistent, qualified batches of cells without the variability associated with isolating fresh neurons each time.
Another option is to derive the cells from induced pluripotent stem (iPS) cells, giving researchers access to cells of human origin. By using the appropriate culture conditions, including patterning molecules such as cytokines and growth factors, for example, the cells can be driven down particular developmental pathways. “The huge advantage of iPS cells is being able to take somatic cell[s] from a patient that has a certain disorder and then generate neurons from them in culture, allowing us to model human neurological disorders,” says Lloyd-Burton. “I just see this field growing enormously.”
But a neuron is a primary cell, and a derived cell that you’re claiming is a neuron is simply a derived cell—you won’t be able to call it a “neuron” in the peer-reviewed literature, says Anthony Krantis, professor of cellular and molecular medicine at the University of Ottawa and president of QBM Cell Science, which supplies the cryopreserved cells for Lonza. There are biochemical markers, morphology and signaling/behavioral criteria “that you have to show and prove before you claim it’s a neuron.”
The long and the short of it
About half of Lloyd-Burton’s customers culture their cells for two weeks or less, which is fine for many biochemical, molecular-biological or early-developmental studies. But “if you want to look at functional, mature neurons as if you’re studying the human adult brain, then you really need to be culturing at least three weeks in vitro,” she says. “At that point, you can see that the cultures have lots of synapses, and if you do electrophysiology you can see that they are talking to each other—there are a lot of spontaneous action potentials going on.” Krantis agrees, recommending that “neuronal cultures (at our optimized density) should mature to 21 days, and ideally 35 to 42 days, in vitro before study.”
Neurons are terminally differentiated, nonproliferating cells, but they will expand in size. Plate the cells on an appropriate substrate; poly-D lysine is the most common, but for added strength in longer-term culture many researchers add an extracellular matrix component like laminin, says Kaur. Cells should be seeded so they are at the appropriate density at the time of the assay. “For electrophysiology, you don’t want your cells too crowded,” she says, “but you want about 90% confluency if you’re harvesting cells for protein expression, so that you collect as many cells as possible.”
These days, cells typically are grown in media such as Life Technologies’ Gibco® Neurobasal® from Thermo Fisher, on a layer of glial cells to provide necessary growth factors. Because glia can take over a culture—especially in the presence of serum—some researchers substitute conditioned medium from glial cells. It’s also common to use a supplement such as Gibco® B-27®—available in different versions without antioxidants or without insulin, for example, or formulated specifically for electrophysiology.
Most major media vendors produce their own versions of Neurobasal and B-27, which Lloyd-Burton calls “variations on a theme … based on the same formulation.” But she notes that raw materials may be differently sourced and may be used at different concentrations, leading to differences among manufacturers, and even between lots. So users may want to test for themselves.
To keep cultures happy, Kaur recommends feeding the cells about every three days by removing half the medium and replacing it with fresh medium. And Krantis points out that maintaining culture osmolarity—by minimizing evaporation and replacing water that has been lost—is crucial to successful long-term culture.
The vast majority of the nearly 4,000 labs culturing neurons obtain them from rodents, says Lloyd-Burton. Others, like Hensley, take advantage of the relative ease and lower cost infrastructure requirements of zebrafish and frogs for short-term studies. But no matter what the organism, preparation, conditions and assays you choose, relax—it’s not like you’re doing brain surgery. Or is it?