In-situ and label-free optical monitoring of the adhesion and spreading of primary monocytes isolated from human blood: Dependence on serum concentration levels

Abstract

Adhesion and spreading of primary monocytes isolated from human blood were monitored utilizing optical waveguide lightmode spectroscopy (OWLS); a highly sensitive label-free biosensor technique using evanescent optical waves generated at a biocompatible surface.Appropriate development on a custom built setup enabled the OWLS cuvette to be operated as a 1.5 ml mini-incubator, controlling both temperature and CO 2 levels. The incubator-equipped OWLS is readily applicable for delicate and long-term studies on sensitive primary cells, demonstrated here through monitoring the serum dependence of the adhesion and spreading of human monocytes. Moreover, the custom-built setup enables the simultaneous monitoring of the position and overall width of the OWLS resonant peaks. This unique feature makes it possible to distinguish the refractive index variations induced by the adsorption of secreted material from refractive index changes provoked by cellular spreading. A definite attachment and spreading activity was observed on the substratum (glassy silica-titania), when the serum level of the culturing medium was 0.0-0.01%.Increasing serum concentration resulted in a steep fall in monocyte surface adhesion and spreading. 1.0% serum level practically abolished all spreading activity measured by OWLS, and the number of attached cells was significantly decreased, too. Serum addition to fully spread cells provoked a reduction in the cell-substratum contact area, clearly detectable by the biosensor. Cell spreading was inhibited by pre-coating the sensor surface with considerable amounts of serum proteins. These findings suggest that monocyte spreading is inhibited by the adsorption of serum biomolecules to the substratum, rather than by soluble factors present in the serum. All of these results were obtained completely non-invasively with real time monitoring; demonstrating the capabilities of OWLS to sensitively monitor the adhesion properties of immune cells isolated from human blood. 3The current study is, therefore, a significant step towards the application of label-free optical biosensors in medical diagnostics.

Incubator proof miniaturized Holomonitor toin situmonitor cancer cells exposed to green tea polyphenol and preosteoblast cells adhering on nanostructured titanate surfaces: validity of the measured parameters and their corrections

“…Our new correction method for estimation of the undetected parts of lamellipodia resulted in more precise evaluations supported by earlier biosensor kinetic data. 13 Measurements with a Holomonitor can be especially useful in combination with other novel label-free biosensing methods 21,22 to obtain a high content analysis of live cell morphology and behavior.…”

Adhesion kinetics of human primary monocytes, dendritic cells, and macrophages: Dynamic cell adhesion measurements with a label-free optical biosensor and their comparison with end-point assays

“…In the past decades, the field has attracted an increasingly broad scientific interest, and the interaction of either monocytes, MFs, or DCs with various proteins (including fibrinogen, 9,24 collagen-I, 25 laminin, 26 serum proteins, 27,28 and biomimetic peptides based on functional sites of fibronectin 29,30 ) have been investigated. However, lacking the appropriate technique, the overwhelming majority of previous studies failed to capture the inherent and essential dynamics of immune cell adhesion, and characterized it only at a single time point.…”

“…Evanescent field-based optical biosensors, including surface plasmon resonance (SPR), 36,37 optical waveguide lightmode spectroscopy (OWLS), 28,[38][39][40] photonic crystal biosensors, 41 grating coupling interferometry, 42,43 and resonant waveguide grating (commonly recognized as Epic) 44,45 biosensors, are considered to be especially straightforward means to monitor surface adhesion. First, they are sensitive only in a $150 nm thick layer closest to the substratum, exactly where establishment and maturation of anchorage and spreading occurs.…”

“…In our former study, we demonstrated the potentials of a first-generation optical biosensor, namely, the OWLS, to measure the adhesion kinetics of primary monocytes in the presence of varying serum concentrations. 28 However, single-channel OWLS lacked the throughput which was necessary to simultaneously measure the adhesion of different types of cells on various surfaces. Thus, here we used the Epic BenchTop (BT), a microplate-based device 45 relying on the same detection principle as OWLS, to study the adhesion kinetics of monocytes isolated from human blood and that of in vitro differentiated, monocytederived macrophages (MDMs), and monocyte-derived dendritic cells (MDCs).…”

Enhanced protein adsorption and cellular adhesion using transparent titanate nanotube thin films made by a simple and inexpensive room temperature process: Application to optical biochips

“…Waveguide sensors were recently exploited in several novel directions; such as the on-line monitoring of oriented protein adsorption [41], extracellular vesicles [42] and human primary immune [16] and cancer [17] cell adhesion in a completely labelfree manner. Here we report on the preparation of TNT coatings on surface-sensitive optical waveguide-based biosensors for quantitative adsorption and adhesion studies.…”