Biophysical Society Thematic Meeting | Singapore

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Mechanobiology of Disease Tuesday Speaker Abstracts
19
A Tale of Two Viscosities: Microviscosity More Important Than Macroviscosity in a
Crowded Microenvironment
Rafi Rashid1,2, Michael Raghunath3,4,5, Thorsten Wohland2,6 .
1Graduate School for Integrative Sciences and Engineering, National University of Singapore,
Singapore, 2Centre for BioImaging Sciences (CBIS), National University of Singapore,
Singapore, 3Department of Biomedical Engineering, National University of Singapore,
Singapore, 4NUS Tissue Engineering Programme, National University of Singapore,
Singapore, 5Department of Biochemistry, National University of Singapore,
Singapore, 6Departments of Biological Sciences & Chemistry, National University of Singapore,
Singapore.
Macromolecular crowders enhance the in vitro differentiation of human mesenchymal stem cells
(hMSCs). The fractional volume occupancy (FVO) is a measure of a polymer’s “excluded
volume”: the higher the FVO, the greater the crowding effects. Based on the FVO of blood
plasma and interstitial fluid proteins, we calculated the optimum physiological FVO needed for
cell culture. However, the existing in vitro FVO (33%) achieved by the carbohydrate polymer,
Ficoll, falls short of the physiological FVO (54%). When we deployed the non-carbohydrate
polymer, polyvinylpyrrolidone (PVP) as an alternative crowder, we could reach 54% FVO and
improve ECM deposition by hMSCs and human fibroblasts. In a collagen fibrillogenesis assay,
PVP accelerated the fibrillogenesis rate over 0 - 54% FVO, whereas Ficoll ceased to enhance the
rate beyond 9% FVO. Bulk viscosity, or macroviscosity, measurements reveal that PVP is less
viscous than Ficoll. Since the rate of a biochemical reaction depends on the positive effect of
excluded volume and the negative effect of macroviscosity, we looked more closely at the effect
of viscosity on reaction kinetics using an in vitro actin polymerization assay. Against
expectations, the actin polymerization rate quadrupled even though the macroviscosity had
increased 60 fold. Glycerol, a pure viscogen, suppressed actin polymerization over the same
macroviscosity range. As suggested by fluorescence correlation spectroscopy (FCS)
measurements, it is the microviscosity, rather than the macroviscosity, that is relevant in a
crowded environment. Our results suggest a model in which a crowder’s excluded volume
increases the reaction rate, but, significantly, the crowder’s microviscosity does not increase
sufficiently to decrease the rate. As our understanding of crowding effects improves, we will be
better able to manipulate in vitro cell culture systems in order to study physiological and
pathological processes.