Application Note 22

Adipose derived mesenchymal stem
cells: Investigating sphere formation in
relation to seeding density and hydrogel
concentration
Jonathan Sheard, Ph.D.
Sheard BioTech Ltd & University of Reading, UK

INTRODUCTION

Culture expansion of cells in 3D versus the classical 2D approach is proving to be aninvaluable tool for use in assay development, for use in drug screening, as well as providing
a clearer understanding of cell behaviour in cell-based regenerative therapies [1,2].
Notably, mesenchymal stem cells (MSCs) are the most researched cells for their potential
use in cell-based therapies. As such, it is crucial to fully characterise these cells withinnovel 3D environments prior to their use and application for such therapies. It has beenpreviously shown that cell seeding density and the 3D cell carrier affect the behaviour anddifferentiation capacity of bone marrow derived and umbilical cord/Wharton’s jelly derived
MSCs towards bone and cartilage regeneration respectively [3, 4, 5].
Here, adipose derived MSCs (ADMSCs) were cultured in nanofibrillar cellulose, GrowDex®,
under various conditions for 1 week to characterise their behaviour ahead of use in thedevelopment of further cell based assays.

MATERIALS• Adipose Derived Mesenchymal Stem Cells (ADMSCs, Cat No. PT-5006, Lonza)• Complete Media: DMEM (High Glucose) supplemented with 10% FBS, 1% Pen/Strepand 1% L-Glutamine (Sigma Aldrich)• 96-well non-treated tissue culture plates (Cat No. 83.3924.500, Sarstedt)• GrowDex, 1.5% (Cat No. 100 103 005, UPM)
• EVOS Live imaging system (ThermoFisher)• Image-J image analysis software (NIH)METHODS

1.
2.
3.

4.
5.
6.
7.

Following 7 d culture expansion of the ADMSCs at 37°C, cell spheres were visible in all
sample wells. The spheres varied in both size and number depending on the initial cellseeding density and the concentration of GrowDex used for the culture.
Small spheres were observed in all wells irrespective of the GrowDex concentration or theinitial cell seeding density used (Fig. 1-2). The number and size of spheres was quantified
for each sample and it was found that the majority of spheres had an average size less than
500 µm2 (Fig. 3). However, in 0.1% GrowDex larger spheres ranging from 500 – 200,000µm2 were also observed at cell seeding densities of 250 – 500 cells/µl. At the highest
cell concentration (1,000 cells/µl) and 0.1% GrowDex a very large sphere, size >200,000µm2, was present in all three wells (Fig.1 C & F)

0.1% GrowDex®

0.1% GrowDex®

50x10325x103

D7100x103

RESULTSAt all times, ADMSCs were cultured in complete media and incubated at 37°C with
10% CO2.
Following trypsinization, cells were resuspended in media at a concentration of 1 x 106
cells/ml.
Cells were then mixed with GrowDex and the appropriate volume of media added
to provide final concerntrations of 0.1%, 0.2% or 0.4% GrowDex seeded with celldensities of 250, 500 or 1000 cells/µl.
100 µl of each of sample were pipetted in triplicate into wells of a 96-well non-tissue
culture plate.
50 µl of media was pipetted carefully on top of the cell/GrowDex mix.
The plate was incubated at 37°C with 10% CO2 with culture media exchange being
performed every 2 to 3 d.
Images of the samples were taken using the EVOS imaging system using 4x and 10x
objectives. Images captured using the 4x objective were loaded into image J where thenumber and area of the cell spheres was measured and quantified.

Figure 1. Primary ADMSCs were seeded at different cell densities within different
concentrations of GrowDex and cultured for 7 d. Cells were seeded at densities of 250, 500
or 1000 cells/µl within GrowDex to a final concentration of 0.1%. Images were captured
using 4x (A-C) and 10x (D-F) magnification.

Sphere formation was observed in the 0.2% GrowDex samples at all cell seeding densities(Fig.2 G-I). However, it was noted that fewer spheres were present in the 0.2% samples andtheir area was smaller when compared to the 0.1% GrowDex samples (Fig.3 A-C).
Within 0.4% GrowDex, the number of spheres were seen to increase relative to the seeding
density, from 250 to 1000 cells/µl. However, these spheres showed little variation in spherearea (Fig.2 J-L, Fig.3 A & D).

0.2% GrowDex®

0.4% GrowDex®

100x10350x10325x103

D7Figure 2. Primary ADMSCs cultured for 7 d in GrowDex at concentrations of 0.2% (G-I)
and 0.4% (J-L) with differing initial cell seeding densities.

Figure 3. Quantification of total cellspheres from images collected in 100µl
of 0.1% GrowDex, 0.2% GrowDex and
0.4% GrowDex (A). Quantification of
the area of the spheres (µm2) observed
in 0.1% GrowDex (B), 0.2% GrowDex(C) and 0.4% GrowDex (D). Cells wereseeded at 250 (top row data), 500(middle row data) and 1000 cells/µl(bottom row data*). *The area of thelargest spheres (>200,000µm2) wereexcluded from the graphs.

CONCLUSIONS

Differences in seeding density is known to impact cell bahaviour. Notably, MSCs isolated
from Wharton’s jellys were shown to have higher glycosaminoglycan and collagen contents
when seeded at densities of 25 to 50 thousand cells/µl on a non-woven polyglycolic acidscaffold [4]. Additionally, bone marrow MSCs seeded onto a copolymer scaffold at higher
cell seeding densities formed more bone than those seeded at lower densities following
osteogenic induction [3].
Aggregated MSC spheres have also been shown to provide enhanced osteogenic andadipogenic differentiation potential [6], as well as produce higher amounts of cartilage
matrix deposition [7]. Additionally, conditioned media harvested from MSCs cultured
as spheres showed stong anti-inflammatory effects [8, 9, 10]. Therefore, it is important tocharacterise the behaviour and potential application of cells embeded within this novel 3D
nanofibrillar cellulose hydrogel, GrowDex.
From the results shown here, it can be easily seen that different cell seeding densities alongwith variable concentrations of GrowDex can affect cell behaviour after 7 days of cellculture; notably the formation cell spheres. However, other affects are yet to be determined.

REFERENCES

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

1.

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