Modification of the swirling well cell culture model to alter shear stress metrics.

Effects of hemodynamic shear stress on endothelial cells have been extensively investigated using the "swirling well" method, in which cells are cultured in dishes or multiwell plates placed on an orbital shaker. A wave rotates around the well, producing complex patterns of shear. The method allows chronic exposure to flow with high throughput at low cost but has two disadvantages: a number of shear stress characteristics change in a broadly similar way from the center to the edge of the well, and cells at one location in the well may release mediators into the medium that affect the behavior of cells at other locations, exposed to different shears. These properties make it challenging to correlate cell properties with shear. The present study investigated simple alterations to ameliorate these issues. Flows were obtained by numerical simulation. Increasing the volume of fluid in the well-altered dimensional but not dimensionless shear metrics. Adding a central cylinder to the base of the well-forced fluid to flow in a square toroidal channel and reduced multidirectionality. Conversely, suspending a cylinder above the base of the well made the flow highly multidirectional. Increasing viscosity in the latter model increased the magnitude of dimensional but not dimensionless metrics. Finally, tilting the well changed the patterns of different wall shear stress metrics in different ways. Collectively, these methods allow similar flows over most of the cells cultured and/or allow the separation of different shear metrics. A combination of the methods overcomes the limitations of the baseline model.

Characterizing nuclear morphology and expression of eNOS in vascular endothelial cells subjected to a continuous range of wall shear stress magnitudes and directionality.

Complex patterns of hemodynamic wall shear stress occur in regions of arterial branching and curvature. Areas within these regions can be highly susceptible to atherosclerosis. Although many studies have characterized the response of vascular endothelial cells to shear stress in a categorical manner, our study herein addresses the need of characterizing endothelial behaviors over a continuous range of shear stress conditions that reflect the extensive variations seen in the vasculature. We evaluated the response of human umbilical vein endothelial cell monolayers to orbital flow at 120, 250, and 350 revolutions per minute (RPM) for 24 and 72 h. The orbital shaker model uniquely provides a continuous range of shear stress conditions from low and multidirectional at the center of each well of a culture plate to high and unidirectional at the periphery. We found distinct patterns of endothelial nuclear area, nuclear major and minor diameters, nuclear aspect ratio, and expression of endothelial nitric oxide synthase over this range of shear conditions and relationships were fit with linear and, where appropriate, power functions. Nuclear area was particularly sensitive with increases in the low and multidirectional WSS region that incrementally decreased as WSS became higher in magnitude and more unidirectional over the radius of the cell layers. The patterns of all endothelial behaviors exhibited high correlations (positive and negative) with metrics of shear stress magnitude and directionality that have been shown to strongly associate with atherosclerosis. Our findings demonstrate the exquisite sensitivity of these endothelial behaviors to incremental changes in shear stress magnitude and directionality, and provide critical quantitation of these relationships for predicting the susceptibility of an arterial segment to diseases such as atherosclerosis, particularly within complex flow environments in the vasculature such as around bifurcations.

Natural Plant Extracts: An Update about Novel Spraying as an Alternative of Chemical Pesticides to Extend the Postharvest Shelf Life of Fruits and Vegetables.

Fresh fruits and vegetables, being the source of important vitamins, minerals, and other plant chemicals, are of boundless importance these days. Although in agriculture, the green revolution was a milestone, it was accompanied by the intensive utilization of chemical pesticides. However, chemical pesticides have hazardous effects on human health and the environment. Therefore, increasingly stimulating toward more eco-friendly and safer alternatives to prevent postharvest losses and lead to improving the shelf life of fresh fruits and vegetables. Proposed alternatives, natural plant extracts, are very promising due to their high efficacy. The plant-based extract is from a natural source and has no or few health concerns. Many researchers have elaborated on the harmful effects of synthetic chemicals on human life. People are now much more aware of safety and health concerns than ever before. In the present review, we discussed the latest research on natural alternatives for chemical synthetic pesticides. Considering that the use of plant-based extracts from aloe vera, lemongrass, or neem is non-chemical by-products of the fruits and vegetable industry, they are proved safe for human health and may be integrated with economic strategies. Such natural plant extracts can be a good alternative to chemical pesticides and preservatives.

Improvement and validation of a computational model of flow in the swirling well cell culture model.

Effects of fluid dynamics on cells are often studied by growing the cells on the base of cylindrical wells or dishes that are swirled on the horizontal platform of an orbital shaker. The swirling culture medium applies a shear stress to the cells that varies in magnitude and directionality from the center to the edge of the vessel. Computational fluid dynamics methods are used to simulate the flow and hence calculate shear stresses at the base of the well. The shear characteristics at each radial location are then compared with cell behavior at the same position. Previous simulations have generally ignored effects of surface tension and wetting, and results have only occasionally been experimentally validated. We investigated whether such idealized simulations are sufficiently accurate, examining a commonly-used swirling well configuration. The breaking wave predicted by earlier simulations was not seen, and the edge-to-center difference in shear magnitude (but not directionality) almost disappeared, when surface tension and wetting were included. Optical measurements of fluid height and velocity agreed well only with the computational model that incorporated surface tension and wetting. These results demonstrate the importance of including accurate fluid properties in computational models of the swirling well method.

Endothelial cells exposed to atheroprotective flow secrete follistatin-like 1 protein which reduces transcytosis and inflammation.

BACKGROUND AND AIMS: When endothelium is cultured in wells swirled on an orbital shaker, cells at the well centre experience putatively atherogenic flow whereas those near the edge experience putatively atheroprotective flow. Transcellular transport is decreased equally in both regions, consistent with it being reduced by a mediator released from cells in one part of the well and mixed in the swirling medium. Similar effects have been inferred for pro-inflammatory changes. Here we identify the mediator and flow characteristics stimulating its release. METHODS AND RESULTS: Medium conditioned by cells swirled at the edge, but not by cells swirled at the centre or cultured under static conditions, significantly reduced transendothelial transport of a low density lipoprotein (LDL)-sized tracer and tumor necrosis factor α (TNF-α)-induced activation and translocation of nuclear factor κB (NF-κB), adhesion molecule expression and monocyte adhesion. Inhibiting transcytosis similarly decreased tracer transport. Unbiased proteomics revealed that cells from the swirled edge secreted substantially more follistatin-like 1 (FSTL1) than cells from the swirled centre or from static wells. Exogenous FSTL1 reduced transport of the LDL-sized tracer and of LDL itself, as well as TNF-α-induced adhesion molecule expression. Bone morphogenetic protein 4 (BMP4) increased transport of the LDL-sized tracer and adhesion molecule expression; FSTL1 abolished these effects. CONCLUSIONS: Putatively atheroprotective flow stimulates secretion of FSTL1 by cultured endothelial cells. FSTL1 reduces transcellular transport of LDL-sized particles and of LDL itself, and inhibits endothelial activation. If this also occurs in vivo, it may account for the atheroprotective nature of such flow.

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies.

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

Endothelial cells do not align with the mean wall shear stress vector.

The alignment of arterial endothelial cells (ECs) with the mean wall shear stress (WSS) vector is the prototypical example of their responsiveness to flow. However, evidence for this behaviour rests on experiments where many WSS metrics had the same orientation or where they were incompletely characterized. In the present study, we tested the phenomenon more rigorously. Aortic ECs were cultured in cylindrical wells on the platform of an orbital shaker. In this system, orientation would differ depending on the WSS metric to which the cells aligned. Variation in flow features and hence in possible orientations was further enhanced by altering the viscosity of the medium. Orientation of endothelial nuclei was compared with WSS characteristics obtained by computational fluid dynamics. At low mean WSS magnitudes, ECs aligned with the modal WSS vector, while at high mean WSS magnitudes they aligned so as to minimize the shear acting across their long axis (transverse WSS). Their failure to align with the mean WSS vector implies that other aspects of endothelial behaviour attributed to this metric require re-examination. The evolution of a mechanism for minimizing transverse WSS is consistent with it having detrimental effects on the cells and with its putative role in atherogenesis.

Single source precursor synthesized CuS nanoparticles for NIR phototherapy of cancer and photodegradation of organic carcinogen.

Herein, we report cost effective and body compatible CuS nanoparticles (NPs) derived from a single source precursor as photothermal agent for healing deep cancer and photocatalytic remediation of organic carcinogens. These NPs efficiently kill MCF7 cells (both in vivo and in vitro) under NIR irradiation by raising the temperature of tumor cells. Such materials can be used for the treatment of deep cancer as they can produce a heating effect using high wavelength and deeply penetrating NIR radiation. Furthermore, CuS NPs under solar light irradiation efficiently convert p-nitrophenol (PNP), an environmental carcinogen, to p-aminophenol (PAP) of pharmaceutical implication. In a nutshell, CuS can be used for the treatment of deep cancer and for the remediation of carcinogenic pollutants. There seems an intrinsic connection between the two functions of CuS NPs that need to be explored in length.

A novel method for segmenting growth of cells in sheared endothelial culture reveals the secretion of an anti-inflammatory mediator.

BACKGROUND: Effects of shear stress on endothelium are important for the normal physiology of blood vessels and are implicated in the pathogenesis of atherosclerosis. They have been extensively studied in vitro. In one paradigm, endothelial cells are cultured in devices that produce spatially varying shear stress profiles, and the local profile is compared with the properties of cells at the same position. A flaw in this class of experiments is that cells exposed to a certain shear profile in one location may release mediators into the medium that alter the behaviour of cells at another location, experiencing different shear, thus obscuring or corrupting the true relation between shear and cell properties. METHODS: Surface coating methods were developed for attaching cells only to some areas of culture-ware and preventing them from spreading into other regions even during prolonged culture. RESULTS: Segmenting the growth of cells had no effect on cell shape, alignment and number per unit area compared to culturing cells in the whole well, but there were differences in tumour-necrosis-factor-α (TNF-α)-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), and monocyte adherence to the monolayer. CONCLUSIONS: The results are consistent with the release of a mediator from cells exposed to high-magnitude uniaxial shear stress that has anti-inflammatory effects on activated endothelium; the mediator may be of importance in atherogenesis. Hence the new methods revealed an important property that would not have been observed without growth segmentation, suggesting that they could find more widespread application.