Differentiation of a Human Neuroblastoma Cell Line into Mature Neurons

Abstract

Source: Shipley, M. M., et al. Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line. J. Vis. Exp. (2016).

This video demonstrates a technique for transforming undifferentiated SH-SY5Y neuroblastoma cells into fully differentiated mature neurons using a systematic passaging technique. This involves culturing the cells in a series of media with gradually decreasing serum levels to inhibit the proliferation of epithelial-like cells while promoting the differentiation and proliferation of neuron-like cells. Additionally, the media is supplemented with retinoic acid and specific growth factors that induce terminal differentiation of neuron-like cells into mature neurons exhibiting an extensive branched morphology.

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 ....

Results

Figure 1: Timetable of differentiation procedure. The differentiation process consists of 11 steps spread out over the course of an 18 day period. On the first day of the differentiation protocol (day 0), between 25,000 and 100,000 cells are plated onto unco.......

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	-