Label-Free Optical Monitoring Of The Adhesion And Spreading Of Human Cells: High Throughput Analysis With Superior Sensitivity And Time Resolution

Here, we briefly discuss the past, present, and possible future of label-free optical biosensors in cell adhesion research. Currently available optical biosensors possess outstanding potentials still not rightfully recognized and still waiting to be fully exploited in the field. Thus, during the description we give special emphasis to the advantages the state-of-the-art optical cell-based biosensors possess as compared to microscope- or force- measurement based techniques that are currently much more generally used to characterize cell adhesion. To name here only a few, they enable label-free detection close to a planar sensor surface, have high sensitivity, and generate superior quality kinetic data. Such information-rich kinetic data, in turn, can be subjected to in-depth comparative and kinetic analysis. To exemplify the importance of in-depth kinetic analysis, we review a recent study, in which the Epic BenchTop high-throughput optical biosensor was used to measure the dependence of adhesion kinetics on the surface density of integrin ligands. Based on the kinetically analyzed data, a model enabling the label-free determination of the dissociation constant for the interaction between adhesion ligands and their native cell membrane receptors has been constructed

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

Monocytes, dendritic cells (DCs), and macrophages (MFs) are closely related immune cells that differ in their main functions. These specific functions are, to a considerable degree, determined by the differences in the adhesion behavior of the cells. To study the inherently and essentially dynamic aspects of the adhesion of monocytes, DCs, and MFs, dynamic cell adhesion assays were performed with a high-throughput label-free optical biosensor [Epic BenchTop (BT)] on surfaces coated with either fibrinogen (Fgn) or the biomimetic copolymer PLL-g-PEG-RGD. Cell adhesion profiles typically reached their maximum at ∼60 min after cell seeding, which was followed by a monotonic signal decrease, indicating gradually weakening cell adhesion. According to the biosensor response, cell types could be ordered by increasing adherence as monocytes, MFs, and DCs. Notably, all three cell types induced a larger biosensor signal on Fgn than on PLL-g-PEG-RGD. To interpret this result, the molecular layers were characterized by further exploiting the potentials of the biosensor: by measuring the adsorption signal induced during the surface coating procedure, the authors could estimate the surface density of adsorbed molecules and, thus, the number of binding sites potentially presented for the adhesion receptors. Surfaces coated with PLL-g-PEG-RGD presented less RGD sites, but was less efficient in promoting cell spreading than those coated with Fgn; hence, other binding sites in Fgn played a more decisive role in determining cell adherence. To support the cell adhesion data obtained with the biosensor, cell adherence on Fgn-coated surfaces 30–60 min after cell seeding was measured with three complementary techniques, i.e., with (1) a fluorescence-based classical adherence assay, (2) a shear flow chamber applying hydrodynamic shear stress to wash cells away, and (3) an automated micropipette using vacuum-generated fluid flow to lift cells up. These techniques confirmed the results obtained with the high-temporal-resolution Epic BT, but could only provide end-point data. In contrast, complex, nonmonotonic cell adhesion kinetics measured by the high-throughput optical biosensor is expected to open a window on the hidden background of the immune cell–extracellular matrix interactions