Application Note 13

3D In Vitro culture of the neuronal cellline SH-SY5Y is sustained in GrowDex®
Joseph Lloyd, Jenna James, Matt Dunn, Daniel Merryweather, Rosemary Fricker, Paul RoachInstitute for Science and Technology in Medicine, Keele University, United Kingdom

INTRODUCTION

Culture of cells in vitro has historically been carried out in two-dimensional (2D) format,
in monolayers or sometimes aggregates/spheroids. In recent years it has now become
widely accepted that cells respond differently to this 2D environment than compared to thethree-dimensional (3D) associations they encounter in normal tissue. Various methods havebeen utilised to expand methods of in vitro culture into 3D, with hydrogel networks being
likely the most widely used. Various in vitro models have been established to replicate
many different organ and tissue types, although the central nervous system remains achallenge due to the many different intercommunicating cell types and the dependence
on complex cytoarchitecture for normal function. The need to develop complex functionalmodels, particularly using human-relevant tissues, will be highly relevant for establishinga better understanding of neurological dysfunction and an enabling technology forclinical intervention. Culturing within a 3D environment, allowing and supporting intercommunication between various neural cell types, is essential in this effort. Here we establish
methodologies for 3D cell culture of the common neuronal cell line SH-SY5Y within theGrowDex® hydrogel to provide a more biomimetic set of environmental cues with which topromote cell migration and neurite outgrowth, whilst sustaining the viability of the population.
We have had similar success with primary cortical (E16) neurons although those details are
not covered in this application note.

MATERIALS
AND METHODS• Initial assays determined that dilution of the hydrogels using media produced asignificantly more stable matrix, resulting in reduced loss of cells during media changes,
fixing and staining when compared to gels diluted using PBS.
• Cells were either pre-mixed with the gel solution else seeded after gelation.
• Each well of a 96-well plate was seeded with 90,000 SH-SY5Y cells.
• Wells were filled with either GrowDex or one of three commonly used peptide hydrogels
for comparison (these are commercially available peptides designed to support neuronalculture) diluted to 1 % w/v with DMEM-F12 cell culture media containing 10% v/v
human serum, 1% L-Glutamine, and 1% pen/strep and allowed to set at 37°C for 24 h.
• Media was changed every second day for seven days to sustain propagation of thepopulation.
• Each well was then either washed with PBS and stained using a live/dead assay or
fixed with formaldehyde and stained using the nuclear stain DAPI and the fluorescentrecombinant anti-β-tubulin antibody E7.

RESULTS

Fluorescence imaging revealed that two of the peptide hydrogels examined had notsupported cell growth owing to dissolution of the hydrogel under the testing regime.
GrowDex and the one remaining peptide gel did retain structure throughout the evaluation
and were shown to support cell growth. GrowDex maintained an evenly distributed cell
population (Fig. 1) which also indicated cells had migrated throughout the hydrogel. The
remaining peptide gel, whilst sustaining high cell viability (Fig. 2), demonstrated minimal
neurite outgrowth compared to those cells cultured in GrowDex.

% Live Cells

Figure 1. Image of SH-SY5Y cells encapsulated within GrowDex stained with DAPI (blue)
and the anti-β-tubulin fluorescent antibody E7 (green), day 7. Scale bar 10 µm.

GrowDex

Peptide gel 3

Figure 2. Live/dead staining of SH-SY5Y cells with 1% v/w GrowDex hydrogel 24 h afterseeding. The results of the assay are compared to the same data generated using the single
peptide hydrogel that was able to maintain the cell culture. Error bars indicate a single
standard deviation.

CONCLUSIONSUPM Biomedicals
Alvar Aallon katu 1P.O. Box 380
00101 Helsinki, Finland
biomedicals@upm.com
www.upmbiomedicals.com
www.upm.com

Our results indicate that a 1% w/v GrowDex hydrogel is able to sustain the three
dimensional culture of SH-SY5Y cells, producing an evenly distributed population within a
96-well plate format. Cell viability was high and neurite outgrowth was also observed inthe 3D format. Additionally it was noticed that the consistency of GrowDex was more robust
when added to media via a syringe rather than vice versa which allowed for more thoroughmixing without the formation of bubbles.

You can order products online at: www.upmbiomedicals.com/store.
Or contact us at biomedicals.sales@upm.com for a quotation or to place an order.