Application Note 15

GrowDex® Supports Robust HumanNeuronal Network Formation in vitro
T. Joki1, L. Ylä-Outinen1, L. Paasonen2, S. Narkilahti1
1
NeuroGroup, BioMediTech, University of Tampere, BioMediTech, Tampere, Finland
2
UPM-Kymmene Oyj, Helsinki, Finland

INTRODUCTION

Human stem cell based neuronal cultures are a promising tool for studying e.g. diseasemechanisms, drug response or developmental biology in vitro. However, traditional flat 2D
cultures on top of a rigid surface fail to reproduce in vivo like brain complexity, like highcell density and connectivity with surrounding cell-cell and cell-extra cellular matrix (ECM)
contacts. Due to these aspects, the 2D culture can lead to unnatural cell polarization on theflat culture. Different types of 3D cell cultures aim to overcome some of these limitations, byoffering cells artificial extracellular matrix (ECM) and more in vivo mimicking environment.
The potential of GrowDex® for 3D culturing of various cell types has already been
demonstrated (Lou et al. 2014; Bhattacharya et al. 2012). It mimics native soft tissue ECM
in fiber size and in mechanical properties, thus providing cells a more in vivo like growthenvironment. In this study pre-differentiated human pluripotent stem cell derived neurons
where encapsulated within GrowDex and cultured.

MATERIALS AND
METHODS

Human embryonic stem cell (hESC) derived neuronal cells encapsulated in GrowDex(UPM) were cultured for two weeks. Studied GrowDex concentrations were 1.50 and 1.0
wt%. Two different hydogel volumes, 80 and 60 μl, were studied using a 96-well plate
format. Human neurons were derived from hESC line Regea 08/023. Briefly, stem cells
were pre-differentiated for eight weeks using previously published method (Lappalainenet al. 2010). The cell concentration used in GrowDex cultures was five million cells per mlof hydrogel. The formation of the neuronal networks inside the hydrogel was evaluatedby immunocytochemical staining against neuronal markers Microtubule-AssociatedProtein 2 (MAP-2) and β-Tubulin III (β-Tub), as previously described (Koivisto et al. 2017).
Immunocytochemical samples were imaged with an Olympus IX51 inverted and Zeiss LSM
780-confocal microscope. Image processing was performed using Adobe Photoshop CS4,
Huygens Essential and ImageJ –softwares. Confocal data was visualised as shown in Fig. 1.

Figure 1. Image processing of theconfocal stacks. Neuronal networks
inside GrowDex were visualizedusing maximum intensity projections(A,B) and 3D rendering (C).

RESULTS

1. GrowDex was a stable 3D matrix
Sample preparation, cell plating, cell culture, and analysis were successful with GrowDex.
The smaller GrowDex volume caused some loss of the material during washes in theimmunocytochemical staining. Overall, the samples were stable during cell culture andanalysis (Fig. 2).

A80 μlSevere gel loss

Moderate gel loss

GrowDex 1.5 wt%0%0%

GrowDex 1.0 wt%0%0%

60 μl

Severe gel loss

Moderate gel loss

GrowDex 1.5 wt%33%0%

GrowDex 1.0 wt%0%33%

B Severe gel loss

C Moderate gel loss

D Intact gel

Figure 2. Samples with gel loss as percentage (A) in both studied GrowDex volumes andfigures describing the classification (B,C,D). Arrow heads show areas of gel loss. Images
were taken after immunocytochemical staining and mounting. Scale bar is 200 μm.

2. GrowDex volume affected cell growth
Neurite outgrowth was analysed from the immunocytochemically stained samples. Cultures
were classified into three categories: 1) robust neurite outgrowth, 2) moderate neurite
outgrowth, or 3) neural aggregates (no outgrowth), according to the amount of visible
neurites (Fig. 3). Smaller hydrogel volume was more supportive of neurite outgrowth whereas
the larger volume supported neural aggregates.

A80 μlRobust neurite
outgrowthModerate neurite
outgrowth

Neural aggregates

GrowDex 1.5 wt%

22%

11%

67%

GrowDex 1.0 wt%

11%

22%

67%

60 μl

Robust neurite
outgrowthModerate neurite
outgrowth

Neural aggregates

GrowDex 1.5 wt%

0%

67%

33%

GrowDex 1.0 wt%

33%

67%

0%

B Robust neurite outgrowth

C Moderate neurite outgrowth D Neural aggregates

Figure 3. Evaluation of neurite outgrowth in 3D cultures (A) and examples of classification(B,C,D). Arrow heads show areas with neurite outgrowth. Scale bar is 200 μm.

NFC 1.5 wt%

3. Robust neurite outgrowth in 3D
Neurite outgrowth was studied in more detail using confocal imaging. Both GrowDex
concentrations supported robust neurite outgrowth in all directions (Fig. 4), in hydrogel
volume of 60 μl.

x: 377.13 μm

y: 355.50 μm

x: 377.13 μm

y: 377.13 μm

x: 377.13 μm

y: 233.50 μm

x: 377.13 μm

NFC 1.0 wt%

y: 377.13 μm

Figure 4. Maximun intensity projections (A,B,D,E) and 3D rendering (C,F) of confocal data(see Fig. 1 for the method). GrowDex concentrations: 1.50 wt% (A,B,C) and 1.0 wt% (D,E,F).
Scale bar is 100 μm.

CONCLUSIONSREFERENCES

Based on the results it can be concluded that GrowDex offers a very good 3D growthenvironment for human neural cells. No clear differences were seen between the two studiedhydrogel concentrations, whereas the hydrogel volume was found to have an unexpected
effect on neurite outgrowth. In larger hydrogel volume (80 μl) the growth as organoid-likeaggregates was favored and smaller hydrogel volume (60 μl) supported neurite outgrowthand cell infiltration. These findings suggest that GrowDex is a promising matrix for 3D humanneural in vitro models.

1.

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4.

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Available at: http://dx.doi.org/10.1016/j.jconrel.2012.06.039
Koivisto, J.T. et al., 2017. Bioamine-crosslinked gellan gum hydrogel for neural tissue
engineering. Biomedical Materials, pp.1–38. Available at: http://iopscience.iop.org/article/10.1088/1748-605X/aa62b0
Lappalainen, R.S. et al., 2010. Similarly derived and cultured hESC lines show
variation in their developmental potential towards neuronal cells in long-term culture.
Regenerative medicine, 5(5), pp.749–762.
Lou, Y.-R. et al., 2014. The Use of Nanofibrillar Cellulose Hydrogel As a Flexible
Three-Dimensional Model to Culture Human Pluripotent Stem Cells. Stem Cells andDevelopment, 23(4), pp.380–392. Available at: http://online.liebertpub.com/doi/abs/10.1089/scd.2013.0314

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