Robert D. Shepherd

Shear stress influences the pluripotency of murine embryonic stem cells in stirred suspension bioreactors

Pluripotent embryonic stem cells (ESCs) have been used increasingly in research as primary material for various tissue‐engineering applications. Pluripotency, or the ability to give rise to all cells of the body, is an important characteristic of ESCs. Traditional methods use leukaemia inhibitory factor (LIF) to maintain murine embryonic stem cell (mESC) pluripotency in static and bioreactor cultures. When LIF is removed from mESCs in static cultures, pluripotency genes are downregulated and the cultures will spontaneously differentiate. Recently we have shown the maintenance of pluripotency gene expression of mESCs in stirred suspension bioreactors during differentiation experiments in the absence of LIF. This is undesired in a differentiation experiment, where the goal is downregulation of pluripotency gene expression and upregulation of gene expression characteristic to the differentiation. Thus, the objective of this study was to examine how effectively different levels of shear stress [100 rpm (6 dyne/cm2), 60 rpm (3 dyne/cm2)] maintained and influenced pluripotency in suspension bioreactors. The pluripotency markers Oct‐4, Nanog, Sox‐2 and Rex‐1 were assessed using gene expression profiles and flow‐cytometry analysis and showed that shear stress does maintain and influence the gene expression of certain pluripotency markers. Some significant differences between the two levels of shear stress were seen and the combination of shear stress and LIF was observed to synergistically increase the expression of certain pluripotency markers. Overall, this study provides a better understanding of the environmental conditions within suspension bioreactors and how these conditions affect the pluripotency of mESCs. Copyright

Methicillin resistant Staphylococcus aureus adhesion to human umbilical vein endothelial cells demonstrates wall shear stress dependent behaviour

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) is an increasingly prevalent pathogen capable of causing severe vascular infections. The goal of this work was to investigate the role of shear stress in early adhesion events.MethodsHuman umbilical vein endothelial cells (HUVEC) were exposed to MRSA for 15-60 minutes and shear stresses of 0-1.2 Pa in a parallel plate flow chamber system. Confocal microscopy stacks were captured and analyzed to assess the number of MRSA. Flow chamber parameters were validated using micro-particle image velocimetry (PIV) and computational fluid dynamics modelling (CFD).ResultsUnder static conditions, MRSA adhered to, and were internalized by, more than 80% of HUVEC at 15 minutes, and almost 100% of the cells at 1 hour. At 30 minutes, there was no change in the percent HUVEC infected between static and low flow (0.24 Pa), but a 15% decrease was seen at 1.2 Pa. The average number of MRSA per HUVEC decreased 22% between static and 0.24 Pa, and 37% between 0.24 Pa and 1.2 Pa. However, when corrected for changes in bacterial concentration near the surface due to flow, bacteria per area was shown to increase at 0.24 Pa compared to static, with a subsequent decline at 1.2 Pa.ConclusionsThis study demonstrates that MRSA adhesion to endothelial cells is strongly influenced by flow conditions and time, and that MSRA adhere in greater numbers to regions of low shear stress. These areas are common in arterial bifurcations, locations also susceptible to generation of atherosclerosis.

Effects of Starch Volume Expanders on Blood Viscosity and Vascular Endothelial Markers

The intravenous fluid of choice for acute blood volume replacement remains controversial. We focus here on the two hydroxyethyl (HES) available in Canada: HES 130/0.40 (Voluven®) and HES 260/0.45 (Pentaspan®). Although information regarding their pharmacokinetic and risk/benefit profiles are available, how the infusion of these fluids could affect blood viscosity and vascular endothelial function in humans is largely unknown. Dynamic viscosity was measured at 21°C and 37°C through capillary viscometry. The HES solutions were driven through a closed flow loop at room temperature (21°C). Viscosity at 21°C was 7.62 centipoise (cP) for HES 260/0.45 and 2.73 cP for HES 130/0.40 decreasing to 4.23 cP for HES 260/0.45 and 1.72 cP for HES 130/0.40 at 37°C. Analysis of viscous behaviour through pipe flow found that HES 260/0.45 displayed marginal variations in viscosity suggesting Newtonian behaviour across our range of Re measured. HES 130/0.40 displayed an appreciable increase in viscosity at higher Re suggesting the presence of shear thickening behaviour. Human aortic endothelial cells (HAEC) and human microvascular endothelial cells (HMVEC) were exposed to the HES solutions and saline to identify chemical effects on vascular endothelium. Western blot quantification showed that E-selectin was the leukocyte adhesion receptor that was most strongly affected, and this was not dose dependent. Interestingly, HAEC and HMVEC had different responses to HES treatment, suggesting that different vascular tissues may have different outcomes to HES infusion. Protein expression in HMVEC decreased when exposed to both HES solutions.Copyright