Harihara Baskaran

Neutrophil chemotaxis in linear and complex gradients of interleukin-8 formed in a microfabricated device.

Although a wealth of knowledge about chemotaxis has accumulated in the past 40 years, these studies have been hampered by the inability of researchers to generate simple linear gradients instantaneously and to maintain them at steady state. Here we describe a device microfabricated by soft lithography and consisting of a network of microfluidic channels that can generate spatially and temporally controlled gradients of chemotactic factors. When human neutrophils are positioned within a microchannel, their migration in simple and complex interleukin-8 (IL-8) gradients can be tested. The cells exhibit strong directional migration toward increasing concentrations of IL-8 in linear gradients. Neutrophil migration halts abruptly when cells encounter a sudden drop in the chemoattractant concentration to zero (“cliff” gradient). When neutrophils are challenged with a gradual increase and decrease in chemoattractant (“hill” gradient), however, the cells traverse the crest of maximum concentration and migrate further before reversing direction. The technique described in this paper provides a robust method to investigate migratory cells under a variety of conditions not accessible to study by earlier techniques.

Analysis of oxygen transport to hepatocytes in a flat-plate microchannel bioreactor.

AbstractOxygen transfer to cultured hepatocytes in microchannel parallel-plate bioreactors with and without an internal membrane oxygenator was investigated with a mathematical model and the results were corroborated with fluorescence imaging experiments. The consumption of oxygen by hepatocytes was assumed to follow Michaelis–Menten kinetics. Our simulations indicate that under conditions of low Péclet (Pe) number (<80) and fixed Damkohler number (=0.14, corresponding to rat hepatocytes) the cells are hypoxic in the bioreactor without an internal membrane oxygenator. Under the same conditions, the bioreactor with an internal membrane oxygenator can avoid cell hypoxia by appropriate selection of membrane Sherwood number and/or the gas phase oxygen partial pressure, thus providing greater control of cell oxygenation. At high Pe, both bioreactors are well oxygenated. Experimentally determined oxygen concentrations within the bioreactors were in good qualitative agreement with model predictions. At low Pe, cell surface oxygen depletion was predicted from analytically derived criteria. Hepatocytes with oxygen dependent functional heterogeneity may exhibit optimal function in the bioreactor with the internal membrane oxygenator.

A method to determine the chemokinetic effect of growth factors using the Fischer equation

We describe a simple method that can be used to determine the chemokinetic coefficient of cells at the boundary of an advancing cell monolayer. The method is based on the use of the Fischer equation and requires only simple measurements of cell proliferation and cell migration. Several samples can be simultaneously studied with reliable and reproducible results using this method.

Poloxamer-188 improves capillary blood flow in the zone of stasis after burn injury

Cutaneous burn injury causes blood flow reduction in regions near the site of the injury, collectively termed as the zone of stasis. Blood flow, in this zone, ceases after 24-48 hours resulting in an expansion of the injured area. The authors used intravital microscopy to investigate the zone of stasis. CD-1 male mice (30 g) were fitted with skin flap chambers and capillary blood flow was visualized by intravenously injecting fluorescently labeled red blood cells into the mice. A temperature-controlled copper tip of 2 mm diameter was used to inflict a full-thickness burn injury on the mouse skin. Real time fluorescent images of the blood flow before and after the burn were recorded. Playback image analysis showed that there was a reduction in blood flow near the site of burn injury immediately (0-2 hrs.) after the injury and away from the site of injury there was no change. Intravenous injection of Poloxamer-188 (0.1 ml, 200 mg/kg) greatly improved blood flow in the zone of stasis.