An Unbiased Flow Cytometry-Based Approach to Assess Subset-Specific Circulating Monocyte Activation and Cytokine Profile in Whole Blood.

Monocytes are the third most frequent type of leukocytes in humans, linking innate and adaptive immunity and are critical drivers in many inflammatory diseases. Based on the differential expression of surface antigens, three monocytic subpopulations have been suggested in humans and two in rats with varying inflammatory and phenotype characteristics. Potential intervention strategies that aim to manipulate these cells require an in-depth understanding of monocyte behavior under different conditions. However, monocytes are highly sensitive to their specific activation state and expression of surface markers, which can change during cell isolation and purification. Thus, there is an urgent need for an unbiased functional analysis of activation in monocyte subtypes, which is not affected by the isolation procedure. Here, we present a flow cytometry-based protocol for evaluating subset-specific activation and cytokine expression of circulating blood monocytes both in humans and rats using small whole blood samples (50 - 100 µL). In contrast to previously described monocyte isolation and flow cytometry visualization methods, the presented approach virtually leaves monocyte subsets in a resting state or fixes them in their current state and allows for an unbiased functional endpoint analysis without prior cell isolation. This protocol is a comprehensive tool for studying differential monocyte regulation in the inflammatory and allogeneic immune response in vitro and vivo.

Mechanical load and mechanical integrity of lung cells - experimental mechanostimulation of epithelial cell- and fibroblast-monolayers.

Experimental mechanostimulation of soft biologic tissue is widely used to investigate cellular responses to mechanical stress or strain. Reactions on mechanostimulation are investigated in terms of morphological changes, inflammatory responses and apoptosis/necrosis induction on a cellular level. In this context, the analysis of the mechanical characteristics of cell-layers might allow to indicate patho-physiological changes in the cell-cell contacts. Recently, we described a device for experimental mechanostimulation that allows simultaneous measurement of the mechanical characteristics of cell-monolayers. Here, we investigated how cultivated lung epithelial cell- and fibroblast-monolayers behave mechanically under different amplitudes of biaxial distension. The cell monolayers were sinusoidally deflected to 5%, 10% or 20% surface gain and their mechanical properties during mechanostimulation were analyzed. With increasing stimulation amplitudes more pronounced reductions of cell junctions were observed. These findings were accompanied by a substantial loss of monolayer rigidity. Pulmonary fibroblast monolayers were initially stiffer but were stronger effected by the mechanostimulation compared to epithelial cell-monolayers. We conclude that, according to their biomechanical function within the pulmonary tissue, epithelial cells and fibroblasts differ with respect to their mechanical characteristics and tolerance of mechanical load.

A method to measure mechanical properties of pulmonary epithelial cell layers.

The lung has a huge inner alveolar surface composed of epithelial cell layers. The knowledge about mechanical properties of lung epithelia is helpful to understand the complex lung mechanics and biomechanical interactions. Methods have been developed to determine mechanical indices (e.g., tissue elasticity) which are both very complex and in need of costly equipment. Therefore, in this study, a mechanostimulator is presented to dynamically stimulate lung epithelial cell monolayers in order to determine their mechanical properties based on a simple mathematical model. First, the method was evaluated by comparison to classical tensile testing using silicone membranes as substitute for biological tissue. Second, human pulmonary epithelial cells (A549 cell line) were grown on flexible silicone membranes and stretched at a defined magnitude. Equal secant moduli were determined in the mechanostimulator and in a conventional tension testing machine (0.49 ± 0.05 MPa and 0.51 ± 0.03 MPa, respectively). The elasticity of the cell monolayer could be calculated by the volume-pressure relationship resulting from inflation of the membrane-cell construct. The secant modulus of the A549 cell layer was calculated as 0.04 ± 0.008 MPa. These findings suggest that the mechanostimulator may represent an adequate device to determine mechanical properties of cell layers.

A new device for dynamic ventilation-analogue mechanostimulation of pliant tissue layers.

The experimental mechanostimulation of biological cell and tissue test samples has become a standard method in biomechanics research. In order to apply a static or a dynamic mechanical load on biological tissue a variety of different devices for the mechanostimulation have been developed. While cyclic load applications are typically restricted to sinusoidal or rectangular stimulation patterns, a device for more complex dynamic stimulation patterns which would simulate, for instance, the dynamics during mechanical ventilation does not exist. The dynamic alveolar recruitment/derecruitment has been identified as one of the main causes of ventilator-induced lung injury. Therefore, there is a demand for an experimental ventilation-analogue mechanostimulation of the pulmonary cells and tissue. Here, we present our mechanostimulator combined with a new driving system which is able to produce the ventilation-analogue patterns of a dynamic mechanostimulation. In an experimental setting where the test samples were simulated by silicone-membranes in single-, double- and fourfold membrane configuration, we varied the stimulation amplitude from 5% to 60% surface increase and stimulation frequencies ranging from 15/min to 2000/min. Furthermore, the frequency components of mechanical load applied to the sample at sinusoidal, rectangular and ventilation- analogue mechanostimulations were analyzed by means of a Fast Fourier Transform (FFT). The system allows for a homogeneous mechanostimulation with various temporal profiles which may include frequency components of up to 20 Hz. The relative amount of mechanical load applied to the sample at the main stimulation frequency was 76% during sinusoidal stimulation, 35% during the rectangular stimulation, and 29% to 42% during ventilation analogue stimulation.