Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

Summary

It is critical in neurobiology and neurovirology to have a reliable, replicable in vitro system that serves as a translational model for what occurs in vivo in human neurons. This protocol describes how to culture and differentiate SH-SY5Y human neuroblastoma cells into viable neurons for use in in vitro applications.

Abstract

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease.

The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods^1-4 and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

Introduction

The ability to use in vitro model systems has greatly enhanced the fields of neurobiology and the neurosciences. Cells in culture provide an efficient platform to characterize protein functionality and molecular mechanisms underlying specific phenomena, to understand the pathology of disease and infection, and to perform preliminary drug testing assessments. In neurobiology, the major types of cell culture models include primary neuronal cultures derived from rats and mice, and neuroblastoma cell lines such as rat B35 cells⁵, Neuro-2A mouse cells⁶, and rat PC12 cells⁷. Although use of such cell lines has advanced the field sig....

Protocol

1. General Considerations

1. See the Table of Materials/Equipment for a list of necessary reagents. Perform all steps under strict aseptic conditions.
2. Use heat-inactivated fetal bovine serum (hiFBS) for all media preparations that include FBS. To heat-inactivate, warm a 50 ml aliquot of FBS at 56 °C for 30 min, inverting every 10 min (see also Table 1).
   Note: When FBS is used without heat-inactivation, the epithelial-like phenotype progresses more quickly throughout cultures of SH-SY5Y cells.
3. Prior to use, allow media to warm and equilibrate in an incubator to establish a proper pH balance befo....

Results

At present, there are many instances in the field of neurobiology and neurovirology where undifferentiated SH-SY5Y cells are being used as a functional model for human neurons^27-36, and importantly, undifferentiated cells may lack phenotypes such as optimal viral uptake² that are necessary for accurate interpretation. It is critical that when using SH-SY5Y cells or any other in vitro neuronal system, cells are appropriately differentiated into neurons, in order to obtain data that is the be.......

Discussion

The above protocol provides a straightforward and reproducible method to generate homogenous and viable human neuronal cultures. This protocol utilizes techniques and practices that integrate several previously published methods^1-4 and aims to delineate the best practices of each. Differentiation of SH-SY5Y cells relies on gradual serum deprivation; the addition of retinoic acid, neurotrophic factors and extracellular matrix proteins; and serial splitting to select for differentiated mature adherent neurons. T.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
B-27	ThermoFisher Scientific	17504-044	See Table 1 for preparation
Brain-Derived Neurotrophic Factor (BDNF)	Sigma	SRP3014 (10 μg)/B3795 (5 μg)	See Table 1 for preparation
dibutyryl cyclic AMP (db-cAMP)	Sigma	D0627	See Table 1 for preparation
DMSO	ATCC	4-X	-
Minimum Essential Medium Eagle (EMEM)	Sigma	M5650	-
Fetal Bovine Serum (FBS)	Hyclone	SH30071.03	See Table 1 for preparation
GlutamaxI	Life Technologies	35050-061	-
Glutamine	Hyclone	SH30034.01	-
Potassium Chloride (KCl)	Fisher Scientific	BP366-1	See Table 1 for preparation
MaxGel Extracellular Matrix (ECM) solution	Sigma	E0282	See step 11 of the protocol
Neurobasal	Life Technologies	21103-049	-
Penicillin/Streptomycin (Pen/Strep)	Life Technologies	15140-122	-
Retinoic acid (RA)	Sigma	R2625	Should be stored in the dark at 4 °C because this reagent is light sensitive
SH-SY5Y Cells	ATCC	CRL-2266	-
0.5% Trypsin + EDTA	Life Technologies	15400-054	-
Falcon 35 mm TC dishes	Falcon (A Corning Brand)	353001	-