Measurement of plaque‐forming macrophages activated by lipopolysaccharide in a micro‐channel chip

Abstract

These findings indicate that LPS-activated macrophages accumulate in micro-channel arrays, and suggest that macrophage plaque formation is a two-step procedure: (1) LPS-activated macrophages adhere physically to the silicon rubber layer on micro-pillars; and (2) consequently, the cells adhere to the activated macrophage layer.

Involvement of adhesion molecule inin vitroplaque-like formation of macrophages stimulated withAggregatibacter actinomycetemcomitanslipopolysaccharide

“…The RAW264.7 cells were cultured in the absence or presence of 1 μg/mL LPS at 37°C for 12 h. The flow test was performed for 0, 20, 60 or 120 min with unstimulated and LPS‐stimulated cells, expressed as LPS (−) and LPS (+), respectively. The side wall of the micropillar was defined as the analysis area (subtracted area from 250 μm × 500 μm to a half area of micropillar; 9.0 × 10 4 μm 2 ; 11). (B) Adhesion rates in unstimulated and LPS‐stimulated RAW264.7 cells.…”

“…We established the method involving the evaluation of in vitro plaque‐like formation by macrophage cells in a fluid system via development of the microchannel chip (11). In the microchannel, plaque‐like formation by RAW264.7 cells increased significantly following LPS stimulation.…”

“…The microchannel chip was fabricated from silicon (Sumitomo 3M, Ltd, Tokyo, Japan) and acrylic resin as described previously (11). In brief, five channels (1.4 mm width, 10 mm length, 200 μm depth) were located on the chip; each channel consisted of 20 micropillars.…”

“…Digital images of macrophages adherent to micropillars in the channels were obtained using a CCD camera connected to the optical microscope. As described in our previous study (11), the side wall of the micropillar, where an impact area with the liquid stream and plaque‐like formation occurred, was defined as the analysis area (9.0 × 10 4 μm 2 ). Areas of plaque‐like formation were measured for 100 analysis areas with image analysis software (DP2‐BSW; Olympus Co., Tokyo, Japan) to calculate the percentage of plaque‐like formation area in each analysis area as an adhesion rate (11).…”

“…We fabricated a microchannel chip to observe both the cell adhesion process and plaque‐like formation (11). We also established a method for the measurement of the difference in adhesion between unstimulated and LPS‐simulated macrophages on a microchannel chip in order to evaluate cell adhesion in a fluid system; this system revealed that LPS‐stimulated macrophages rapidly adhere following a short incubation period in a flowing liquid stream.…”