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.
Highlights
SH-SY5Y cells are a human neuroblastoma-derived cell line used to model human neurons in vitro
We investigated the effects of serum removal on cytoskeletal markers of neuron morphology by visualizing changes in MAP2 and βIII-Tubulin
We found that complete serum removal has a specific effect on the expression of several genes involved in BDNF-TrkB signaling and synaptogenesis
Summary
SH-SY5Y cells are a human neuroblastoma-derived cell line used to model human neurons in vitro. The original cells were derived from a bone marrow biopsy in 1970, and were cloned to produce the neuron-like SH-SY5Y cells that are used in a wide range of research applications today (Biedler et al 1978). SH-SY5Y cells express a catecholaminergic phenotype, with the potential to synthesize both dopamine and noradrenaline (Krishna et al 2014) They can be used to study synapse modifications and functional cellular activity with live calcium imaging or electrophysiology (Santillo et al 2014; Toselli et al 1996; Jahn et al 2017). They are often used as a cell model for Parkinson’s Disease (Xicoy et al 2017), as well as Alzheimer’s Disease (Agholme et al 2010), neuropathogenesis of viruses (Christensen et al 2011), screening for neurotropic properties of pharmaceuticals (Henkel et al 2008; Xu et al 2019), neurotoxicity (De Simone et al 2018; Forster et al 2016), and even as a multicellular 3D culture (Cui et al 2017; Kapalczynska et al 2018)
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