Application Note 23

Hanging drop culture ofmesenchymal stem cells
within nanofibrillar cellulose
Jonathan Sheard, Ph.D.
Sheard BioTech Ltd & University of Reading, UK

INTRODUCTION

Mesenchymal stem cells (MSCs) are clinically relevant, tripotent adult stem cells that giverise to bone, cartilage and fat cells. MSCs can be efficiently isolated from various sources
including bone marrow, fat and many dental tissues. In vitro, MSCs can be cultivated asconventional 2D monolayer cultures and, as reported recently [1], within nanofibrillar
cellulose (GrowDex®). It has also been suggested, that cultivation of MSCs as 3D spheroids
might increase their anti-inflammatory potential [2,3]. Briefly, MSCs cultured within hanging
drops begin to aggregate and form spheres within 3 days [4] and these spheres remain
viable. However, the number of apoptotic or necrotic cells may increase relative to a higher
seeding density of cells or culture time [2]. Additionally, MSCs cultured as spheroids show
higher differentiation potential towards cartilage regeneration when cultured within 3D
scaffolds or as hanging drop spheres [5,6].
This application note describes an easy method for generation of MSC-spheres using
GrowDex®. Utilising a 3D matrix such as GrowDex in the hanging drop technology may
provide a useful tool for examining; cell-cell and cell-matrix behaviour, in addition to celldifferentiation potential, within smaller sample volumes and with fewer cells.

MATERIALS• Adipose Derived Mesenchymal Stem Cells (ADMSCs, Cat# PT-5006, Lonza)• Complete Media: DMEM (High Glucose) supplemented with 10% FBS, 1% Pen/Strepand 1% L-Glutamine (Sigma Aldrich)• Non-treated tissue culture dishes - 60x15mm (Cat# 83.3901.500, Sarstedt)• GrowDex, 1.5% (Cat# 100 103 005, UPM)
• EVOS Live imaging system (ThermoFisher)• Image-J image analysis software (NIH)METHODS

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Through 7 days of culture expansion, ADMSC spheres were visible within all hanging
drop samples (Fig.1). The spheres varied in both size and number depending on the theconcentration of GrowDex used for the culture (Fig.2).
Small spheres were observed in all hanging drops irrespective of the GrowDex concentration
used (Fig.1). The number and size of spheres was quantified for each hanging drop (Fig.2).
At 7 days of culture, GrowDex was observed to condense within the hanging drop (Fig.1C, G). It was also noted, at the lower concentration of 0.2% (Fig.1 C), GrowDex did not
maintain a regular pellet shape compared to that of the higher concentration of 0.4% (Fig.1G).
At day 1, an average of 55 cell spheres were counted in 0.2% GrowDex hanging drops(Fig.1 A-B, Fig.2 A), with an area of 124 µm2 per sphere (Fig.2 B). After 7 days of culture,
the number of spheres increased approximately 2 fold to 116 (Fig 2. A) and the area also
increased approximately 2 fold to 250 µm2 per sphere (Fig.2 B).
In 0.4% GrowDex an average of 38 cell spheres per hanging drop where counted on day
1 which increased to 77 spheres per hanging drop by day 7 (Fig.2 A). The area of eachsphere at day 1 was approximately 133 µm2, increasing only 1.5 fold to 203 µm2 per cellsphere.

Day 70.2% GrowDex®

Day 10.4% GrowDex®

RESULTSADMSCs 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.
Subsequently, cells were mixed with the appropriate volume of media and GrowDex toprovide final concentrations of 0.2% and 0.4% GrowDex seeded with 100 cells/µl.
A droplet of 10 µl per sample was pipetted onto the lid of a non-tissue culture treatedpetri-dish. No less than 3 droplets were prepared per sample.
Petri-dishes were incubated at 37°C with 10% CO2.
5 µl of complete culture media was added every 1 to 2 days.
Images were taken using the EVOS imaging system using 4x and 10x objectives.
Images captured using the 10x objective were loaded into image J where the number
and area of the cell spheres was measured and quantified.

Figure 1. Images showing primary ADMSCs cultured as hanging drops through 7 days
using GrowDex concentrations of 0.2% (A-D) and 0.4% (E-H). Images were captured at 4x(A, C, E, G) and 10x (B, D, F, H) magnification.

Figure 2. Quantification of the average number of cell spheres per hanging drop (A) andthe area (µm2) per sphere from images collected of droplets at day 1 (B) and day 7 (C).

CONCLUSIONS

Embedding cells within a 3D matrix and culturing these as hanging drops, enables
the observation and measurement of both spatial cell-cell interactions and cell-matrix
interactions within a small volume [7]. Combining multiple cell types as a co-culture system
can also provide further opportunity to closely mimic in vivo conditions [8]. Additionally,retrieval of the spheres from hanging drop cultures has been detailed and is quite simple [4].
MSC spheres cultured within hanging drops have been shown to posses enhanced
osteogenic and adipogenic differentiation potential [9], and produce higher amounts ofcartilage matrix deposition [6]. Moreover, conditioned media harvested from MSCs spheres
has been shown to have stong anti-inflammatory effects [2, 3, 4].
Here, an alternative method of culturing MSC spheres within GrowDex has been
demonstrated. The results show that MSCs can be easily embedded in GrowDex and,following 7 days of culture, these MSCs form small compact cell spheres which increase insize and number. This method offers a useful alternative approach to study cell behavioiur,interaction and differentiation potential, with the added benefit of requiring smaller sample
volumes and fewer cells.

REFERENCES

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Azoidis, I., J., et al. (2017). "Three-dimensional cell culture of human mesenchymal stem
cells in nanofibrillar cellulose hydrogels." MRS Communications: 1-8.
Bartosh, T. J., et al. (2010). "Aggregation of human mesenchymal stromal cells (MSCs)
into 3D spheroids enhances their anti-inflammatory properties." Proceedings of theNational Academy of Sciences of the United States of America 107(31): 1372413729.
Ylöstalo, J. H., et al. (2012). "Human mesenchymal stem/stromal cells (hMSCs)
cultured as spheroids are self-activated to produce prostaglandin E2 (PGE2) that directs
stimulated macrophages into an anti-inflammatory phenotype." Stem cells (Dayton,Ohio) 30(10): 2283-2296.
Bartosh, T. J. and J. H. Ylostalo (2014). "Preparation of anti-inflammatory mesenchymal
stem/precursor cells (MSCs) through sphere formation using hanging drop culturetechnique." Current protocols in stem cell biology 28: Unit-2B.6.
Wang, L., et al. (2009). "Effect of Initial Seeding Density on Human Umbilical Cord
Mesenchymal Stromal Cells for Fibrocartilage Tissue Engineering." Tissue Engineering.
Part A 15(5): 1009-1017.
Suzuki, S., et al. (2012). "Properties and usefulness of aggregates of synovial
mesenchymal stem cells as a source for cartilage regeneration." Arthritis Research &Therapy 14(3): R136-R136.

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Matak, D., et al. (2017). "Colony, hanging drop, and methylcellulose three dimensional
hypoxic growth optimization of renal cell carcinoma cell lines." Cytotechnology 69(4):565-578.
Edmondson, R., et al. (2014). "Three-Dimensional Cell Culture Systems and Their
Applications in Drug Discovery and Cell-Based Biosensors." Assay and Drug
Development Technologies 12(4): 207-218.
Frith, J. E., et al. (2010). "Dynamic three-dimensional culture methods enhancemesenchymal stem cell properties and increase therapeutic potential." Tissue Eng Part C
Methods 16(4): 735-749.

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