The Effects of Serum Removal on Gene Expression and Morphological Plasticity Markers in Differentiated SH-SY5Y Cells

Abstract

Despite the widespread use of the SH-SY5Y human neuroblastoma cell line in modeling human neurons in vitro, protocols for growth, differentiation and experimentation differ considerably across the literature. Many studies fully differentiate SH-SY5Y cells before experimentation, to investigate plasticity measures in a mature, human neuronal-like cell model. Prior to experimentation, serum is often removed from cell culture media, to arrest the cell growth cycle and synchronize cells. However, the exact effect of this serum removal before experimentation on mature, differentiated SH-SY5Y cells has not yet been described. In studies using differentiated SH-SY5Y cells, any effect of serum removal on plasticity markers may influence results. The aim of the current study was to systematically characterize, in differentiated, neuronal-like SH-SY5Y cells, the potentially confounding effects of complete serum removal in terms of morphological and gene expression markers of plasticity. We measured changes in commonly used morphological markers and in genes related to neuroplasticity and synaptogenesis, particularly in the BDNF-TrkB signaling pathway. We found that complete serum removal from already differentiated SH-SY5Y cells increases neurite length, neurite branching, and the proportion of cells with a primary neurite, as well as proportion of βIII-Tubulin and MAP2 expressing cells. Gene expression results also indicate increased expression of PSD95 and NTRK2 expression 24 h after serum removal. We conclude that serum deprivation in differentiated SH-SY5Y cells affects morphology and gene expression and can potentially confound plasticity-related outcome measures, having significant implications for experimental design in studies using differentiated SH-SY5Y cells as a model of human neurons.

Lipid levels correlate with neuronal and dopaminergic markers during the differentiation of SH-SY5Y cells

“…PRKCA) in BRT-differentiated SH-SY5Y cells compared to undifferentiated cells, as reported previously for RA-, RA/BDNF- or RA/TPA-differentiated cells 49,71,74 . Finally, we found that both BMP4 pre-treatment and the removal of serum during the RA and/or TPA led to a more pronounced increase in neurite length, as described previously for serum removal 75 , and more pronounced changes in the levels of lipid species associated with SH-SY5Y cells differentiation.…”

Optimised techniques for high-throughput screening of differentiated SH-SY5Y cells and application for neurite outgrowth assays

“…A number of studies have differentiated the SH-SY5Y cell line with RA for a short duration of 2–4 days, at which point the cells are then treated with a chemical factor, to evaluate the inhibitory or stimulatory effect on neurite outgrowth over a specified period 13 , 29 , 30 . However, such short differentiation protocols would be insufficient to produce a population of terminally differentiated cells 31 . Hence, their use is unlikely to capture the behaviour of mature neurons 28 .…”

Organotypic spinal cord cultures: An <em>in vitro</em> 3D model to preliminary screen treatments for spinal muscular atrophy

“… 12 Moreover in presence of C2C12 conditioned media, we observed a progressive centrifugal migration of the spinal cord cells, possibly in the attempt to arrange in a monolayer in search to a better access to nutrients present in the C2C12-medium, compared to the multilayer disposition in the slice. Lastly, we cannot exclude that the presence of FBS in the culture medium could partially affect the morphological changes we observed 37 as well as its eventual deprivation: 38 however, the significant differences between the treated and untreated samples confirm the prevailing and effective action of the conditioned media.…”