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 significantly, there are several confounding factors associated with handling non-human cells and tissue. These include understanding species-specific differences in metabolic processes, phenotypes of disease manifestation, and pathogenesis when compared to humans. It is also important to note that there are significant differences between mouse and human gene expression and transcription factor signaling, highlighting the limitations of rodent models and the importance of understanding which pathways are conserved between rodents and humans^8-11. Others have employed the use of human neuronal cell lines including the N-Tera-2 (NT2) human teratocarcinoma cell line and inducible pluripotent stem cells (iPSCs). These cell lines provide good models for in vitro human systems. However, differentiation of NT2 cells with retinoic acid (RA) results in the generation of a mixed population of neurons, astrocytes, and radial glial cells¹², necessitating an additional purification step to obtain pure populations of neurons. Additionally, NT2 cells demonstrate a highly variable karyotype¹³, with greater than 60 chromosomes in 72% of cells. iPSCs demonstrate variability in differentiation between different cell lines and vary in differentiation efficiency¹⁴. It is therefore desirable to have a consistent and reproducible human neuronal cell model to complement these alternatives.

SH-SY5Y neuroblast-like cells are a subclone of the parental neuroblastoma cell line SK-N-SH. The parental cell line was generated in 1970 from a bone marrow biopsy that contains both neuroblast-like and epithelial-like cells¹⁵. SH-SY5Y cells have a stable karyotype consisting of 47 chromosomes, and can be differentiated from a neuroblast-like state into mature human neurons through a variety of different mechanisms including the use of RA, phorbol esters, and specific neurotrophins such as brain-derived neurotrophic factor (BDNF). Prior evidence suggests that the use of different methods can select for specific neuron subtypes such as adrenergic, cholinergic, and dopaminergic neurons^16,17. This latter aspect makes SH-SY5Y cells useful for a multitude of neurobiology experiments.

Several studies have noted important differences between SH-SY5Y cells in their undifferentiated and differentiated states. When SH-SY5Y cells are undifferentiated, they rapidly proliferate and appear to be non-polarized, with very few, short processes. They often grow in clumps and express markers indicative of immature neurons^18,19. When differentiated, these cells extend long, branched processes, decrease in proliferation, and in some cases polarize^2,18. Fully differentiated SH-SY5Y cells have been previously demonstrated to express a variety of different markers of mature neurons including growth-associated protein (GAP-43), neuronal nuclei (NeuN), synaptophysin (SYN), synaptic vesicle protein II (SV2), neuron specific enolase (NSE) and microtubule associated protein (MAP)^2,16,17,20, and to lack expression of glial markers such as glial fibrillary acidic protein (GFAP)⁴. In further support that differentiated SH-SY5Y cells represent a homogeneous neuronal population, removal of BDNF results in cellular apoptosis⁴. This suggests that survival of differentiated SH-SY5Y cells is dependent on trophic factors, similar to mature neurons.

Use of SH-SY5Y cells has increased since the subclone was established in 1978³. Some examples of their use include investigating Parkinson's disease¹⁷, Alzheimer's disease²¹, and the pathogenesis of viral infection including poliovirus²², enterovirus 71 (EV71)^23,24, varicella-zoster virus (VZV)¹, human cytomegalovirus²⁵, and herpes simplex virus (HSV)^2,26. It is important to note that several studies using SH-SY5Y cells have used these cells in their undifferentiated form, especially in the field of neurovirology^27-36. The difference in the observed phenotype of undifferentiated versus differentiated SH-SY5Y cells raises the question of whether the observed progression of infection would be different in mature differentiated neurons. For example, differentiated SH-SY5Y cells have a higher efficiency of HSV-1 uptake versus undifferentiated, proliferating SH-SY5Y cells, which may be due to a lack of surface receptors that bind HSV and modulate entry on undifferentiated SH-SY5Y cells². It is therefore critical that when designing an experiment focused on testing neurons in vitro, SH-SY5Y cells should be differentiated in order to obtain the most accurate results for translation and comparison to in vivo models.

The development of a reliable method to generate human neuronal cultures is imperative to allow researchers to perform translational experiments that accurately model the human nervous system. The protocol presented here is a procedure that delineates best practices derived from previous methods^1-4 to enrich for human neurons that are differentiated using retinoic acid.

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 before every step. For example, 50 ml of media takes approximately one hour to fully equilibrate (pH 7, 37 °C, 5% CO₂).
   Note: This protocol uses a two-step splitting procedure that requires partially differentiated SH-SY5Y cells to be trypsinized and re-plated. This is a stressful process for these exceptionally fragile cells. Therefore, it is important to incubate the cells in trypsin for a minimal amount of time. This will allow for the preferential lift-off of neurons, leaving epithelial-like cells still attached to the dish.
4. Perform trituration of differentiated cells slowly with a 10 ml plastic pipet with the tip against the bottom of the conical tube containing the cells. Perform trituration at a slow speed, up and down no more than five times.

2. Passage of SH-SY5Y Maintenance Cultures

1. Split maintenance cultures when cells have reached 70-80% confluency, and do not exceed 10 to 15 passages. Cultures typically need to be passaged every 3-5 days (assuming cultures are not diluted more than 5-fold during splitting).
2. To passage cells from a T-75 flask, aspirate off media, then rinse with approximately 10 ml 1x PBS.
   Note: We do not recommend splitting the SH-SY5Y maintenance cultures further than 1:5 during passaging because this can cause the cells to die due to low confluency.
3. Aspirate PBS, and then add 2.5 ml 0.05% Trypsin-EDTA (1x).
4. Incubate in incubator 2-3 min and tip gently to release cells from the surface of the flask.
5. Add 10 ml Basic Growth Media (see Table 2) and triturate 1-2 times.
6. Spin down for 2 min at 1,000 x g, aspirate media, then resuspend pellet in 5 ml Basic Growth Media.
7. Dilute cells from 1:3 to 1:5 in a total volume of 20 ml for normal plating in T-75 flask, or count and plate for differentiation (section 5).

3. Freezing SH-SY5Y Cells

1. Freeze early passages of SH-SY5Y neuroblastoma cells in Basic Growth Media supplemented with 5% (v/v) DMSO.
2. Initially, freeze aliquots at -80 °C for 24 hr, then transfer to liquid nitrogen for long-term storage.
   Note: For reference, a confluent (75-85%) T-75 flask will yield five 1 ml aliquots of SH-SY5Y cells for freezing. Each of these aliquots should contain anywhere from 2-5 million cells total.

4. Thaw and Culture Undifferentiated SH-SY5Y Neuroblastoma Cells

1. Prepare Basic Growth Media.
2. Rapidly thaw frozen cells in a 37 °C water bath (approximately 2 min).
3. Resuspend cells in 9 ml Basic Growth Media in a 15 ml conical tube, and then centrifuge for 2 min at 1,000 x g.
4. Aspirate the supernatant while being careful not to disturb the pelleted cells, and gently resuspend cells in 10 ml Basic Growth Media.
5. Plate cells onto a T-25 flask or 60 mm² dish.
6. The next day, replace media to remove dead cells.

5. Day 0: Plating Cells for Differentiation

1. See Figure 1 for the differentiation schedule.
2. Rinse undifferentiated cells with 1x PBS, aspirate, and then trypsinize using 1-2 ml warmed 1x 0.05% Trypsin-EDTA.
3. When cells are in trypsin, incubate for approximately 3 min in an incubator.
4. Quench the trypsin by adding 10 ml Basic Growth Media, rinse the sides of the flask or dish, and gently triturate 1-3 times. Transfer contents to a 15 ml conical tube.
5. Centrifuge for 2 min at 1,000 x g, and aspirate the media while being careful not to disturb the pellet.
6. Resuspend pellet in 5 ml Basic Growth Media and triturate 1-3 times.
7. Count cells using a hemocytometer, then dilute using Basic Growth Media to 50,000 cells/ml.
8. Plate 2 ml of cells per 35 mm² dish for a total of 100,000 cells per dish and place back into incubator.

6. Day 1: Change Media (Differentiation Media #1)

1. Aliquot 50 ml of Differentiation Media #1 (see Table 2)and incubate in a 37 °C water bath.
2. When media is warmed, allow it to equilibrate in an incubator (37 °C, 5% CO₂) for at least one hr to establish a proper pH balance prior to use.
3. Add Retinoic Acid (RA) (see Table 1) to warmed and equilibrated media immediately before adding media to dishes.
   Note: Retinoic acid is light sensitive and should be stored in dark bottles at 4 °C
4. Gently aspirate off old media and discard.
5. Add 2 ml Differentiation Media #1 with RA per 35 mm² dish and return to incubator.

7. Day 3: Change Media (Differentiation Media #1)

1. Repeat Section 6 (steps 1-5)

8. Day 5: Change Media (Differentiation Media #1)

1. Repeat Section 6 (steps 1-5)

9. Day 7: Split Cells 1:1

1. Add RA to warmed and equilibrated Differentiation Media #1 immediately before adding media to dishes.
2. Gently aspirate off old media and discard.
3. Add 200 µl warmed 0.05% 1x Trypsin EDTA per 35 mm² dish and warm in incubator approximately 2-3 min or until cells are visibly lifted from the plate as observed under a microscope.
4. Quench the trypsin by adding 2 ml Differentiation Media #1 with RA per 35 mm² dish and use the media to rinse remaining neuronal cells off the plate. Then transfer contents to a 50 ml conical tube.
   Note: During trypsinization steps, do not trypsinize too many dishes at once. This helps to ensure neuronal cultures are not incubated in trypsin for too long, which can be cytotoxic.
5. Combine contents from up to 10 dishes in the 50 ml conical tube and gently triturate slowly up and down no more than five times with a 10 ml plastic pipet.
6. Aliquot 2 ml cell suspension into fresh 35 mm² dishes and return to incubator.

10. Day 8: Change Media (Differentiation Media #2)

1. Add RA (see Table 1) to warmed and equilibrated media immediately before adding media to dishes.
2. Gently aspirate off old media and discard.
3. Slowly add 2 mL Differentiation Media #2 (see Table 2) with RA per 35 mm² dish and return to incubator. Do not allow neurons to be exposed to air for an extended period of time as they can dry out quickly.

11. Day 9: Prepare Extracellular Matrix (ECM) Coated Dishes

1. Thaw one vial of ECM solution on ice and dilute 1:100 into cold DMEM.
2. Dispense 2 ml of mixture into each 35 mm² dish and ensure the entire base of the dish is covered.
3. Place in an incubator (37 °C, 5% CO₂) for 1 hr or overnight.
4. Aspirate mixture and allow to air dry for approximately 1 hr in a hood. Store at room temperature for up to 2 months.

12. Day 10: Transfer Cells onto ECM Coated Plates 1:1

1. Add RA (see Table 1) to warmed and equilibrated media immediately before adding media to dishes.
2. Gently aspirate off media and discard.
3. Add 200 µl warmed trypsin to each 35 mm² dish and allow to incubate at room temperature for approximately 1-2 min or until neurons are visibly lifted from the dish as observed under a microscope.
   Note: Execute this trypsinization step at room temperature so as not to over-incubate neurons with trypsin and cause damage. Neurons release from plates much faster than epithelial-like cells at this stage.
4. Quench the trypsin by adding 2 ml Differentiation Media #2 per 35 mm² dish and use the media to rinse remaining neuronal cells off the plate. Then transfer contents to a 50 ml conical tube.
5. Combine contents from up to 10 dishes in the 50 ml conical tube and gently triturate slowly up and down no more than five times with a 10 ml plastic pipet.
6. Dispense 2 ml cell suspension into ECM-coated 35 mm² dishes and return to incubator.

13. Day 11: Change Media (Differentiation Media #3)

1. Add RA (see Table 1) to warmed and equilibrated media immediately before adding media to dishes.
2. Gently aspirate off old media and discard.
3. Slowly add 2 ml Differentiation Media #3 (see Table 2) with RA per 35 mm² dish and return to incubator. Do not allow neurons to be exposed to air for an extended period of time.

14. Day 14: Change Media (Differentiation Media #3)

1. Repeat Section 13 (steps 1-3)

15. Day 17: Last Media Change (Differentiation Media #3)

1. Repeat Section 13 (steps 1-3)

16. Day 18: Neuronal Cultures Ready to Use

1. Change media to fresh Differentiation Media #3 with RA every 3 days to maintain neuron health.
   Note: Cells should be differentiated into neurons and exhibit a neuronal phenotype. Cultures are typically stable for up to 14 days following terminal differentiation, however duration of neuron viability is dependent on passage number of the undifferentiated cells at the start of differentiation. Higher passage numbers yield differentiated neurons with a shorter useful lifetime.

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

Name	Company	Catalog Number	Comments
B-27	Invitrogen	17504-044	See Table 1 for preparation
Brain-Derived Neurotrophic Factor (BDNF)	Sigma	SRP3014 (10ug)/B3795 (5ug)	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 35mm TC dishes	Falcon (A Corning Brand)	353001	-