In patients with peripheral sensory neuron pathologies, including Friedreich ataxia (FA), using the 50B11 cell line as a model for various applications in neuroscience has been suggested, according to findings from a morphological investigation published in the International Journal of Molecular Sciences.
It is well known that evaluation of the physiology and signaling of sensory neurons is key to treating a variety of disorders that involve the peripheric nervous system, such as FA, Fabry disease, porphyria, Charcot-Marie-Tooth, Guillain-Barre syndrome, and type 1 diabetes. In prior years, investigators have used primary dorsal root ganglion (DRG) neurons from rodents, which is a time-consuming, ethically debatable process.
An alternative method to dissociated DRG neurons is immortalized cell lines, such as those generated from the rat cell line 50B11. As an immortalized rat DRG sensory neuronal cell line, upon its differentiation with the plant metabolite forskolin, 50B11 attains typical sensory neuron morphology with extended neurites and markers of small-diameter nociceptive neurons. In this way, 50B11 can serve as a favorable model for mimicking nociceptive sensory neurons in vitro.
During the process of culturing the 50B11 cell line, the researchers noted that 50B11 cells exhibited the ability to grow and differentiate (when treated with forskolin) in conventional conditions, as well as when the cells were permitted to grow on the underside of cover glasses that were deposited in multiwell culture plates.
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In the past, 50B11 has been used to conduct in vitro studies on the effect of analgesic treatment, neurotoxicity, neuronal regeneration, and viral infection. To analyze this unique cell differentiation type in 50B11 cells, the researchers used label-free, nonlinear microscopy to monitor the various differentiations and growths of 50B11 cell lines on the upper and bottom sides of glass substrates, comparing them at 24, 48, and 72 hours.
Via analysis of the cells on the topside and the underside of the glass substrates of the same wells, they monitored the growth of more than 3 preparations of 50B11 cells after the addition of forskolin. Second harmonic generation (SHG) microscopy was used to delineate the cell border and specificity of the SHG signals on cellular structures, thus monitoring cell differentiation.
Even though a clear difference was not observed between the cells growing on the 2 sides of the cover glasses, the morphology of the cells that grew on the topside seemed to include a higher proportion with rounded bodies and short neurites, with a progressive rise in differentiating cell number over the time course. In contrast, in the same well, those cells growing on the underside seemed to differentiate earlier, rapidly generating a highly enlarged neural network.
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In both cell populations growing on the underside and the topside of the cover glasses, a strong SHG signal was reported over the time course of 24, 48, and 72 hours. The multiphoton autofluorescence of the cells was observed as well, which was used to identify the cells and to confirm whether those being analyzed were viable and functional.
“This work represents the first instance of examining neuronal cell lines growing and differentiating in an upside-down orientation, allowing a possible improvement of 50B11 as a model in physiology studies of sensory neurons in peripheric nervous system disease . . . and analgesic drug screening,” the researchers concluded.
Reference
Zupin L, Psilodimitrakopoulos S, Celsi F, et al. Upside-down preference in the forskolin-induced in vitro differentiation of 50B11 sensory neurons: a morphological investigation by label-free non-linear microscopy. Int J Mol Sci. Published online May 6, 2023. doi:10.3390/ijms24098354
