Biophysical Society Thematic Meeting | Singapore

Mechanobiology of Disease

Tuesday Speaker Abstracts

A Tale of Two Viscosities: Microviscosity More Important Than Macroviscosity in a Crowded Microenvironment Rafi Rashid 1,2 , Michael Raghunath 3,4,5 , Thorsten Wohland 2,6 . 1 Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore, 2 Centre for BioImaging Sciences (CBIS), National University of Singapore, Singapore, 3 Department of Biomedical Engineering, National University of Singapore, Singapore, 4 NUS Tissue Engineering Programme, National University of Singapore, Singapore, 5 Department of Biochemistry, National University of Singapore, Singapore, 6 Departments 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.

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