Application Note 10

Bone marrow derived mesenchymal
stem cells cultured in 3D matrix
Chennakesava Cuddapah, Ph.D.
CELLnTEC Advanced Cell Systems AG, Switzerland

INTRODUCTION

Bone marrow mesenchymal stem cells (BM-MSCs), also referred to as BM mesenchymal
stromal cells or marrow stromal cells, are self-renewing and expandable stem cells. The BMMSCs are present as a rare cell population in the bone marrow, representing about 0.001%
to 0.01% of the nucleated cells present. They are approximately 10-fold less abundant
than hematopoietic stem cells (HSCs), are expandable in culture, multipotent and capableof differentiating into several cell types. Numerous animal studies suggest that BM-MSCs
contribute to the repair/regeneration of a variety of injured tissues including the myocardium,
cardiac valves, bone, tendon, cartilage, and meniscus making them a suitable candidate touse for orthopedic tissue engineering and regenerative medicine.
In this study, we investigated the suitability of GrowDex® hydrogel as a matrix for 3D cultureof human bone marrow-derived mesenchymal stem cells (hBM-MSC). The potential for cells
to form spheroids was observed over a 7 day period and cell viability determined using
AlamarBlue.

MATERIALS• HMSC-BM, Human Mesenchymal Stem Cells, Bone Marrow (Cat No. FC-0057, LifelineCell Technology)• StemLife™ MSC-BM Medium Complete Kit (Cat No. LL-0062, Lifeline Cell Technology)• GA Antimicrobial Supplement, 0.5 mL (Cat No. LS-1104, Lifeline Cell Technology)• GrowDex 1.5% (Cat No. 100 100 105, UPM)
• Low-attachment 96-well plate (Corning)

METHODS

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BM-MSCs were maintained according to Lifeline Cell Technology’s protocol which canbe found at https://www.lifelinecelltech.com.
Cells were amplified, collected, then mixed with GrowDex and cell culture media toprovide a final GrowDex concentration of 0.5% and cell density of 1 million cells/ml.
65 µl of GrowDex/cell mix was dispensed into each well of a 96-well microplate andthen 65 µl of culture medium carefully dispensed on top.
The microplate was incubated at 37°C and culture medium replaced daily.
Cell viability was determined by adding 1/10 of the total volume (medium + hydrogel)of AlamarBlue to all wells and the plate shaken for 10 min at room temperature.

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RESULTS

The plate was then incubated for 3 h at 37°C in 5 % CO2 after which it was shaken fora further 10 min at room temperature.
50 μl of medium from each culture well was transferred to another 96-well plate.
The fluorescent signal measured using a plate reader with an excitation wavelength of
480 nm and emission wavelength of 520 nm.

BM-MSCs were able to quickly form spheres and establish cell function. Already after 1 hfrom seeding it was observed that the cells started to migrate in GrowDex to form spheroids(Fig. 1A). By day 1, several spheroids were evident, having an average diameter of 140 µm(Fig 1B). Images from day 7 showed that the spheroids were starting to disaggregate (Fig.
1C). Cell viability was evaluated with AlamarBlue assay over the 7 d culture. The cells in 2D
demonstrated an increased mitochondrial activity through the 7 d culture, whereas the cells inGrowDex showed an increased activity till day 1 and declined thereafter (Fig. 2).

ABC

Figure 1. Human bone marrow –derived mesenchymal stem cells in GrowDex A) after 1 hfrom seeding, B) after 24 h, and c) after 7 d culture.

Figure 2. Cell viability (metabolic activity) determined using AlamarBlue assay studied over
7 d in 2D culture and GrowDex 3D culture.

CONCLUSIONSREFERENCES

Mesenchymal stem cells (MSCs) hold great promise for regenerative therapies and tissueengineering applications. Here we have demonstrated that GrowDex is a suitable matrix
for 3D culture of BM-MSCs with spheroids forming quickly after 24 h in culture. The results
suggest that the optimal period to use BM-MSC spheroids in GrowDex is 3-4 d afterseeding.

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Stoltz et al. 2015. Stem Cells and Regenerative Medicine: Myth or Reality of the 21thCentury. Stem Cells Int. 2015;2015:734731
Rampersad 2012. Multiple Applications of Alamar Blue as an Indicator of Metabolic
Function and Cellular Health in Cell Viability Bioassays. Sensors (Basel) 12(9): 12347–12360

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