Embryonic stem cells (ESCs) can differentiate into all somatic cell types, but the development of effective strategies to direct ESC fate is dependent upon defining environmental parameters capable of influencing cell phenotype. ESCs are commonly differentiated via cell aggregates referred to as embryoid bodies (EBs), but current culture methods, such as hanging drop and static suspension, yield relatively few or heterogeneous populations of EBs. Alternatively, rotary orbital suspension culture enhances EB formation efficiency, cell yield, and homogeneity without adversely affecting differentiation. Thus, the objective of this study was to systematically examine the effects of hydrodynamic conditions created by rotary orbital shaking on EB formation, structure, and differentiation. Mouse ESCs introduced to suspension culture at a range of rotary orbital speeds (20-60 rpm) exhibited variable EB formation sizes and yields due to differences in the kinetics of cell aggregation. Computational fluid dynamic analyses indicated that rotary orbital shaking generated relatively uniform and mild shear stresses (< or =2.5 dyn/cm(2)) within the regions EBs occupied in culture dishes, at each of the orbital speeds examined. The hydrodynamic conditions modulated EB structure, indicated by differences in the cellular organization and morphology of the spheroids. Compared to static culture, exposure to hydrodynamic conditions significantly altered the gene expression profile of EBs. Moreover, varying rotary orbital speeds differentially modulated the kinetic profile of gene expression and relative percentages of differentiated cell types. Overall, this study demonstrates that manipulation of hydrodynamic environments modulates ESC differentiation, thus providing a novel, scalable approach to integrate into the development of directed stem cell differentiation strategies.

Objective-The goal of this study was to examine the effect of chronic heterogeneous shear stress, applied using an orbital shaker, on endothelial cell morphology and the expression of cyclooxygenases 1 and 2. Methods and Results-Porcine aortic endothelial cells were plated on fibronectin-coated Transwell plates. Cells were cultured for up to 7 days either under static conditions or on an orbital shaker that generated a wave of medium inducing shear stress over the cells. Cells were fixed and stained for the endothelial surface marker CD31 or cyclooxygenases 1 and 2. En face confocal microscopy and scanning ion conductance microscopy were used to show that endothelial cells were randomly oriented at the center of the well, aligned with shear stress nearer the periphery, and expressed cyclooxygenase-1 under all conditions. Lipopolysaccharide induced cyclooxygenase-2 and the production of 6-ketoprostaglandin F 1␣ in all cells. Key Words: eicosanoids Ⅲ endothelium Ⅲ prostacyclin Ⅲ cyclooxygenase Ⅲ shear stress E ndothelial cells line the luminal surface of blood vessels and are continuously exposed to hemodynamic shear stress. The level of shear stress that cells experience varies from region to region within the vasculature. In areas of high laminar shear stress, endothelial cells are elongated, aligned, and protected from inflammation. In areas of low, oscillatory shear stress, endothelial cells are randomly orientated and susceptible to inflammation. Areas of low shear stress are thought to be atheroprone, whereas areas of high shear stress are thought to be atheroprotected. [1][2][3] Cultured endothelial cells are routinely studied under static conditions, where they appear nonaligned, with a cobblestone morphology. 4,5 It is increasingly recognized that endothelial cells grown under static conditions may not be representative of endothelial cells in the body. 6,7 In addition, evidence suggests that endothelial endocrine function and expression of key enzymes, including cyclooxygenase (COX), is also regulated by shear stress. 8 COX is present in 2 isoforms: COX-1 and COX-2. Generally, COX-1 is expressed constitutively, whereas COX-2 is induced at sites of inflammation. 9 In endothelial cells, COX-1 activity results predominantly in the production of the antithrombotic hormone prostacyclin. 10 COX-1 and COX-2 are the targets for nonsteroidal antiinflammatory drugs (NSAIDs). They have attracted much media attention since the association of COX-2-selective NSAIDs with adverse cardiovascular events, 11,12 although the mechanism behind this association remains unclear. One leading hypothesis is that COX-2-selective drugs reduce the production of the cardioprotective hormone prostacyclin, 13,14 which, in susceptible individuals, increases the risk of arterial thrombosis. Prostacyclin is formed mainly by endothelial cells, which express high levels of COX. It has previously been shown that COX-1 predominates over COX-2 in endothelial cells cultured under static conditions, 15-17 which raises the question of how COX-2-s...

Endothelial properties are affected by mechanical stresses. Several studies have shown that an acute application of shear stress increases the permeability of endothelial monolayers in culture. We investigated whether more prolonged application of shear has the opposite effect. Porcine aortic endothelial cells were cultured on Transwell filters to assess monolayer permeability to albumin. The medium above the cells was swirled using an orbital shaker; resultant shears were computed to lie within the physiological range. Acute application of shear increased permeability, but chronic application reduced it. The effect of chronic but not acute shear was reversed by inhibiting nitric oxide (NO) synthesis. The effect of chronic shear was also reversed by inhibiting phosphatidylinositol 3-OH kinase (PI3K) and soluble guanylyl cyclase. None of these interventions affected permeability under static conditions, and inhibition of cyclooxygenase was without effect. Chronic shear decreased mitosis rates by a fraction comparable to the reduction in permeability, but this effect was not reversed by inhibiting NO synthesis. We conclude that chronic application of shear stress reduces endothelial permeability to macromolecules by a PI3K-NO-cGMP-dependent mechanism. Since atherosclerosis can be triggered by excessive entry of plasma macromolecules into the arterial wall, the phenomenon may help explain the atheroprotective effects of shear and NO.

Wall shear stress (WSS) on anchored cells affects their responses, including cell proliferation and morphology. In this study, the effects of the directionality of pulsatile WSS on endothelial cell proliferation and morphology were investigated for cells grown in a Petri dish orbiting on a shaker platform. Time and location dependent WSS was determined by computational fluid dynamics (CFD). At low orbital speed (50 rpm), WSS was shown to be uniform (0-1 dyne/cm(2)) across the bottom of the dish, while at higher orbital speed (100 and 150 rpm), WSS remained fairly uniform near the center and fluctuated significantly (0-9 dyne/cm(2)) near the side walls of the dish. Since WSS on the bottom of the dish is two-dimensional, a new directional oscillatory shear index (DOSI) was developed to quantify the directionality of oscillating shear. DOSI approached zero for biaxial oscillatory shear of equal magnitudes near the center and approached one for uniaxial pulsatile shear near the wall, where large tangential WSS dominated a much smaller radial component. Near the center (low DOSI), more, smaller and less elongated cells grew, whereas larger cells with greater elongation were observed in the more uniaxial oscillatory shear (high DOSI) near the periphery of the dish. Further, cells aligned with the direction of the largest component of shear but were randomly oriented in low magnitude biaxial shear. Statistical analyses of the individual and interacting effects of multiple factors (DOSI, shear magnitudes and orbital speeds) showed that DOSI significantly affected all the responses, indicating that directionality is an important determinant of cellular responses.