Validation of a Three-Dimensional Culture System for the Differentiation of Multipotential Mesenchymal Stromal Cells by Uniaxial Strain

The differentiation potential of multipotential mesenchymal stromal cells is known to be
affected by many aspects of the cellular microenvironment, including soluble factors,
extracellular matrix composition, the Young’s modulus of the substrate, cellular neighbours
and externally applied forces. Despite this, reasonable understanding of harnessing soluble
factors only exists. Few studies have investigated mechanotransduction in hMSC, and those
published to date primarily employ unsuitable substrates, that do not facilitate the cellular
adhesions known to be active in force transmission.
In this study, porcine pericardium was decellularised for use as a biologically-relevant, threedimensional
scaffold for the mechanostimulation of hMSC in a uniaxial strain bioreactor.
Tissue stocks (n=67) were successfully decellularised and confirm biocompatible, sterile and
free of contaminating genomic DNA. Histoarchitecture comparable to that of native tissue was
also maintained. Tencell-specific seeding rings were found to release cytotoxic residue, and an
alternative, nontoxic seeding approach was developed.
The Tencell bioreactor was initially unable to maintain cell viability as a culture system, and
was validated with respect to chamber humidity, culture temperature and arm displacement.
Temperature maintenance was inadequate prior to re-engineering of the heating apparatus
and was rectified through the use of an autotunable module. Losses of cell viability were still
observed following validation as a result of medium pH changes. A Tencell culture regime
utilising the HEPES buffer was successfully developed for the culture of hMSC.
No significant differences in gene expression between strained and unstrained samples were
found, and the greatest effects were observed between unseeded and other sample types.
Additionally, seeded hMSC did not penetrate the scaffold.
Overall, this study investigated the differentiation potential of hMSC cultured in a threedimensional
scaffold. The Tencell bioreactor was fully validated for use as a uniaxial strain
mechanostimulation device, and could be used in future studies to investigate the effect of
different frequencies and magnitudes of cyclic strain.