Application Note 1

Flexible 3D model for the cultivation ofhuman embryonic andinduced pluripotent stem cells
Yan-Ru Lou (Ph.D.)
University of Helsinki, Finland
INTRODUCTION

In recent years there has been a drive to move away from the traditional 2D cell culture
systems to what is considered the more physiologically relevant three dimensional (3D)
models. It is thought that cells cultured in 3D more closely mimic the in vivo cell niche that
supports cell proliferation. Here we present a 3D model that has been developed for theculture of human embryonic stem cells (hESC) and induced pluripotent stem cells (iPCS)
where WA07 and H9-GFP (modified from H9) hESC lines and iPCS(IMR90)-4 cells wereused for the study. Cells were cultured in GrowDex® for 9 days in a 96-well plate format,
where cell proliferation rates and spheroid formation was observed and reported. Spheroids
provide a useful 3D model for studying disease states such as cancer and offer a simple touse platform for drug discovery and regenerative medicine research. Developing the assay
in a 96-well plate format allows easy transfer to automated systems for scale up and higher
throughput as required for high throughput screening (HTS).

MATERIALS•••••

H9- GFP modified from H9 (WA09, WiCell) WA07 and iPS(IMR90)-4 (WiCell)mTeSR1 medium (STEMCELL Technologies)
GrowDex 1.5% (UPM)
1mg/ml dispase solution (STEMCELL Technologies)
Live/dead viability/cytotoxicity kit (Invitrogen)

METHODS

1.
2.

GrowDex hydrogel (1.5%) was diluted with culture medium to 0.5% concentration.
Cells previously seeded and cultured in 2D were detached from the culture vessel bynon-enzymatic cell dissociation reagent and collected by centrifugation at 200g for 5min.
The supernatant was removed and the pellet re-suspended carefully in culture medium.
The hESCs were seeded as small colonies, not as single cells or small groups of cells.
The small colonies were generated by passing them through 1000 µl pipette tip
approximately 10 times.
Cell seeding density used for 3D culture in GrowDex was five times higher than that of
the 2D culture before passage. The appropriate number of colonies were mixed withGrowDex by careful pipette transfer and slow mixing with the pipette tip in the solution
to ensure an even distribution of the colonies in the gel.
100 µl of cell/GrowDex mix was transferred to each well of the 96-well plate and 100µl of culture medium added on top of cell/GrowDex mix carefully to ensure the gel was
not disturbed.

3.

4.

5.

METHODS contd.

6.
7.

RESULTS

Cells were maintained for 9 days at 37°C with 5% CO2 in a humid atmosphere.
Culture medium was renewed daily.
Cell viability was assessed using the Live/Dead viability/cytotoxicity kit according
to manufacturer’s instructions and spheroid formation was observed by standard
microscopy.

Here, we propose a new concept of hPSC culture, which is a flexible, xeno-free 3D invitro culture system. We have demonstrated that cells can be successfully cultured, cell
viability retained and spheroids formed ranging in size from 100-200 nm. Unlike mostpolysaccharide-based hydrogels that require a separate cross-linking step, GrowDex can beused following simple dilution with media. Additionally, hydrogels generated by cross-linking
have fixed physical properties, whereas the physical property of GrowDex is dependent on
its concentration determined by dilution, not on the degree of crosslinking. Phase-contrast
images show that cell density and spheroid diameter increased with culture time. The cells in
3D spheroids remained alive during the culture as shown in live/dead viability assays at theend of subcultures.

Fig 1. Human embryonic stem cells(hESCs) and induced pluripotent stem
cells (iPSCs) form three-dimensional(3D) spheroids in GrowDex. A timecourse shows that WA07 (A) andiPS(IMR90)-4 (B) cells formed 3Dspheroids in 0.5% GrowDex. Live/deadstaining images show living WA07
(C) and iPS(IMR90)-4 (D) cells in thespheroids on day 9. Scale bars = 200
nm. Reproduced with permission from(1). Copyright 2014, Mary Ann Liebert,
Inc.

CONCLUSIONSREFERENCE

This study shows that GrowDex can offer a flexible, xeno-free 3D model for culture ofhESC and iPS cells and opens up a new avenue for better recapitulation of natural ESC
environment and embryonic development. This has not been possible with conventional 2D
cultures methods previously.
1.

Lou Y-R et al. 2014 “The Use of Nanofibrillar Cellulose Hydrogel As a Flexible ThreeDimensional Model to Culture Human Pluripotent Stem Cells. STEM CELLS AND
DEVELOPMENT 23:4, pp. 380-392.

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.

www.upm.com

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