The SH-SY5Y cell line is one of the most widely used cell models for studying neurodegenerative diseases. In this post, we provide details on how to utilize the SH-SY5Y cell line for research purposes, including protocols for successful gene editing and cell culture.

Introduction to SH-SY5Y Cell Line Applications

In recent years, SH-SY5Y cells have been widely used as an in vitro model in studies related to immunology, neurodegenerative diseases and neurotoxicity. Data indicate that high levels of brain cholesterol can promote the accumulation of β-amyloid (Aβ) and oxidative stress. SH-SY5Y serves as a model for brain disorders since the addition of specific compounds can convert cells into various types of functional neurons. In one study, to further explore the underlying mechanisms, researchers used SH-SY5Y cells to verify the effects of a high-cholesterol environment on neuronal cells. [1]

Image source:《Translational neurodegeneration》

（https://doi.org/10.1186/s40035-023-00343-3）

What Are SH-SY5Y Cells?

Cell Line Model Name: SH-SY5Y (Human Neuroblastoma Cells)  

Catalog Number: ATCC CRL-2266  

Growth Characteristics: Grows in clusters or aggregates, with a small portion remaining in suspension  

Culture Conditions: MEM/F12 + 10% FBS + Glutamine + NEAA + Sodium Pyruvate  

Culture Environment: 95% air + 5% CO₂; 37°C


The SH-SY5Y cell line is a subline that originated from the third clone of the neuroblastoma cell line SK-N-SH (ATCC HTB-11) (SK-N-SH → SH-SY → SH-SY5 → SH-SY5Y). SK-N-SH was first established in 1970 from a metastatic bone tumor in a 4-year-old cancer patient. SH-SY5Y cells are one of the most widely used cell models for studying neurodegenerative diseases because mature neurons do not divide, while SH-SY5Y cells are capable of continuous proliferation. As undifferentiated cells. exhibiting neuroblast-like morphology and expressing immature neuronal markers. Genetically edited SH-SY5Y cell lines are becoming increasingly important tools in pharmacology and neuroscience research.

Applications of SH-SY5Y Cells

➢ Neurodegenerative Disease Research

Alzheimer's Disease: Used to study the toxic effects of β-amyloid (Aβ) and Tau protein accumulation on neurons.

Parkinson's Disease: Employed to investigate the function of dopaminergic neurons and the aggregation of α-synuclein.

➢ Neurotoxicity Research

Assessment of the toxic effects of various chemicals, drugs, and environmental toxins on neurons.

➢ Neuronal Differentiation

SH-SY5Y cells can be induced to differentiate into neuron-like cells using retinoic acid (RA) or other chemical inducers, making them a valuable model for studying the mechanisms of neuronal differentiation and maturation.

➢ Gene Function

Study the role of specific genes in neuronal function and disease through techniques such as gene knockout, overexpression, or RNA interference.

➢ Signal Transduction

Investigate neuronal signal transduction pathways, such as calcium signaling, the MAPK/ERK pathway, and the PI3K/Akt pathway.

SH-SY5Y Cell Culture Methods

In practice, SH-SY5Y cells are relatively sensitive and proliferate slowly, making it challenging to prepare single clones. To ensure the stability of SH-SY5Y cells and the reliability of experimental results, it is essential to master effective cell culture techniques that facilitate the unique needs of this cell line model.

Cell Culture Procedure

1.  Cell Thawing

  • Add 6 mL of complete culture medium to a centrifuge tube to start the thawing process.
  • Thaw the cells in a water bath until only a small ice pellet remains, then stop the water bath.
  • Transfer the cell suspension to the centrifuge tube and centrifuge.
  • Resuspend the cells and seed them into an appropriately sized culture dish.
  • After 24 hours, check for cell adhesion and change the medium once.


2. Cell Passaging

  • Aspirate & discard the supernatant, and rinse the cells with room temperature PBS once.
  • Add trypsin to evenly cover the bottom of the dish.
  • Digest until some cells begin to detach when tapping the dish, then stop the digestion.
  • Gently pipette to resuspend the cells and transfer them to a centrifuge tube for centrifugation.
  • Resuspend the cells in fresh medium and seed them at the desired ratio.


3. Cell Cryopreservation

  • Digest and centrifuge the cells to obtain a cell pellet.
  • Resuspend the cells in cryopreservation solution and transfer them to cryovials.
  • Place the cryovials into a pre-cooled (4°C) controlled-rate freezing container, then store them overnight in a -80°C freezer.
  • Transfer the cryovials to liquid nitrogen storage the following day.

General Cell Culture Precautions

  • Passage Ratio: Avoid using a high passage ratio and low culture density. Do not over-pass the cells or culture them at too low a density; a 1:2 or 1:3 passaging ratio is optimal. The lower the culture density, the slower the proliferation rate.
  • Overgrowth: When cells overgrow, they tend to clump together and gradually detach. It's essential to passage the cells before they begin to detach.
  • Cell Clumps: During passaging, some cell clumps may need to be dispersed. Be sure to apply moderate force when pipetting.
  • Culture Medium: The higher the cell density, the faster the culture medium is consumed. Ensure timely medium changes or passaging to maintain healthy cell growth.

Gene Editing of SH-SY5Y Cell Line Using Smart-CRISPR™

Gene Modification Projects

Common genetic modifications in the SH-SY5Y cell line include knockout (KO), point mutation (PM), knock-in (KI), overexpression (OE), and interference. Cyagen has optimized preparation and single-cell cloning conditions to deliver monoclonal gene-edited SH-SY5Y cell line models, significantly enhancing the accuracy and reliability of experiments. This approach provides a solid foundation for neuroscience research, enabling more precise investigations into neuronal functions and disease mechanisms.

➢ Gene Knockout

Based on the Smart-CRISPR™ cell gene editing system, Cyagen offers custom SH-SY5Y KO (knockout) cell services, delivering single clone homozygous lines. We utilize an optimized transfection system to directly deliver RNP (ribonucleoprotein) into cells, achieving gRNA cutting efficiency exceeding 90%. Compared to plasmid and virus-mediated CRISPR/Cas methods, this approach significantly increases cutting efficiency while markedly reducing off-target effects, enabling more precise cutting of target DNA sequences.

➢ Point Mutation

Using the Smart-CRISPR™ cell gene editing system, we offer precise and efficient SH-SY5Y point mutation and knock-in (KI) cell customization services. Through an optimized α-donor system, humanized Cas proteins, gRNA, and donor DNA are co-transfected into the target cells, resulting in high-efficiency homologous recombination with an HDR (homology-directed repair) rate of up to 49%. This enables the delivery of single clone homozygous lines, providing a robust tool for advanced research.

➢ Stable Cell Line

Cyagen has optimized and upgraded the transfection vector system, leveraging years of expertise in cell biology to establish a mature and stable gene overexpression experimental system. We offer SH-SY5Y stable cell pools or single clone cell lines that stably express exogenous genes or RNA interference elements, achieving over a 10-fold increase in protein expression. This provides researchers with reliable tools for consistent gene expression studies.

Gene Editing Precautions

  • Cell Preparation: Ensure that cells are in good condition before transfection, with a cell density of around 70%. Under the microscope, avoid having too many cell clumps.
  • Cell Dissociation: During digestion, try to disperse the cells into single cells as much as possible.
  • Single Clone Preparation: Use the limiting dilution method to prepare single clones. Utilize Cyagen's custom culture medium, adding an extra 10% serum and appropriate cytokines. When clones are observed, replenish the medium with fresh culture medium and continue cultivation.
  • Clone Expansion: Since SH-SY5Y cells proliferate slowly at low confluency, it is recommended to expand single clones gradually—first from a 96-well plate to a 24-well plate, then to a 12-well plate, and finally to a 6-well plate. Avoid transferring the cells directly into a large dish for expansion.

Custom Cell Line Gene Editing Services by Cyagen

Cyagen offers a one-stop in vitro service platform for cell line/iPSC gene editing, featuring advanced cell reprogramming, genetic engineering, and cellular differentiation technologies. By integrating our one-stop phenotypic analysis platform, we can provide in vitro model development and testing services for cell lines across various disease applications. Our optimized CRISPR-Pro technology enables rapid gene editing, including the excision of  large fragments, large fragment knockin, and point mutations with stable expression across various cell lines.

Contact our experts and take advantage of our optimized CRISPR-Pro gene edited cell line modeling service platform for low-cost cell line generation with rapid turnaround and stable expression.

>>Learn more about our custom cell line gene editing services

 

References:
[1]de Dios, Cristina., de Dios, Cristina., de Dios, Cristina., Abadin, Xenia.,  and Roca-Agujetas, Vicente.. "Inflammasome activation under high cholesterol load triggers a protective microglial phenotype while promoting neuronal pyroptosis." Translational neurodegeneration.

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