Area and Geometry Dependence of Cell Migration in Asymmetric Two-State Micropatterns.

Microstructured surfaces provide a unique framework to probe cell migration and cytoskeletal dynamics in a standardized manner. Here, we report on the steady-state occupancy probability of cells in asymmetric two-state microstructures that consist of two fibronectin-coated adhesion sites connected by a thin guidance cue. In these dumbbell-like structures, cells transition between the two sites in a repeated and stochastic manner, and average dwell times in the respective microenvironments are determined from the cell trajectories. We study the dynamics of human breast carcinoma cells (MDA-MB-231) in these microstructures as a function of area, shape, and orientation of the adhesion sites. On square adhesive sites with different areas, we find that the occupancy probability ratio is directly proportional to the ratio of corresponding adhesion site areas. These asymmetries are well captured by a simple model for the stochastic nonlinear dynamics of the cells, which reveals generic features of the motion. Sites of equal area but different shape lead to equal occupancy if shapes are isotropic (e.g., squared or circular). In contrast, an asymmetry in the occupancy is induced by anisotropic shapes like rhombi, triangles, or rectangles that enable motion in the direction perpendicular to the transition axis. Analysis of the two-dimensional motion of cells between two rectangles with orthogonal orientation suggests that cellular transition rates depend on the cell polarization induced by anisotropic micropatterns. Taken together, our results illustrate how two-state micropatterns provide a dynamic migration assay with distinct dwell times and relative cell occupancy as readouts, which may be useful to probe cell-microenvironment interactions.

A high-throughput microscopy method for single-cell analysis of event-time correlations in nanoparticle-induced cell death.

The temporal context of cell death decisions remains generally hidden in ensemble measurements with endpoint readouts. Here, we describe a method to extract event times from fluorescence time traces of cell death-related markers in automated live-cell imaging on single-cell arrays (LISCA) using epithelial A549 lung and Huh7 liver cancer cells as a model system. In pairwise marker combinations, we assess the chronological sequence and delay times of the events lysosomal membrane permeabilization, mitochondrial outer membrane permeabilization and oxidative burst after exposure to 58 nm amino-functionalized polystyrene nanoparticles (PS-NH2 nanoparticles). From two-dimensional event-time scatter plots we infer a lysosomal signal pathway at a low dose of nanoparticles (25 µg mL-1) for both cell lines, while at a higher dose (100 µg mL-1) a mitochondrial pathway coexists in A549 cells, but not in Huh7. In general, event-time correlations provide detailed insights into heterogeneity and interdependencies in signal transmission pathways.

Microscopy-based high-throughput assays enable multi-parametric analysis to assess adverse effects of nanomaterials in various cell lines.

Manufactured nanomaterials (MNMs) selected from a library of over 120 different MNMs with varied compositions, sizes, and surface coatings were tested by four different laboratories for toxicity by high-throughput/-content (HT/C) techniques. The selected particles comprise 14 MNMs composed of CeO2, Ag, TiO2, ZnO and SiO2 with different coatings and surface characteristics at varying concentrations. The MNMs were tested in different mammalian cell lines at concentrations between 0.5 and 250 µg/mL to link physical-chemical properties to multiple adverse effects. The cell lines are derived from relevant organs such as liver, lung, colon and the immune system. Endpoints such as viable cell count, cell membrane permeability, apoptotic cell death, mitochondrial membrane potential, lysosomal acidification and steatosis have been studied. Soluble MNMs, Ag and ZnO, were toxic in all cell types. TiO2 and SiO2 MNMs also triggered toxicity in some, but not all, cell types and the cell type-specific effects were influenced by the specific coating and surface modification. CeO2 MNMs were nearly ineffective in our test systems. Differentiated liver cells appear to be most sensitive to MNMs, Whereas most of the investigated MNMs showed no acute toxicity, it became clear that some show adverse effects dependent on the assay and cell line. Hence, it is advised that future nanosafety studies utilise a multi-parametric approach such as HT/C screening to avoid missing signs of toxicity. Furthermore, some of the cell type-specific effects should be followed up in more detail and might also provide an incentive to address potential adverse effects in vivo in the relevant organ.

Time-Resolved Study of Nanoparticle Induced Apoptosis Using Microfabricated Single Cell Arrays.

Cell fate decisions like apoptosis are heterogeneously implemented within a cell population and, consequently, the population response is recognized as sum of many individual dynamic events. Here, we report on the use of micro-patterned single-cell arrays for real-time tracking of nanoparticle-induced (NP) cell death in sets of thousands of cells in parallel. Annexin (pSIVA) and propidium iodide (PI), two fluorescent indicators of apoptosis, are simultaneously monitored after exposure to functionalized polystyrene (PS - NH 2) nanobeads as a model system. We find that the distribution of Annexin onset times shifts to later times and broadens as a function of decreasing NP dose. We discuss the mean time-to-death as a function of dose, and show how the EC 50 value depends both on dose and time of measurement. In addition, the correlations between the early and late apoptotic markers indicate a systematic shift from apoptotic towards necrotic cell death during the course of the experiment. Thus, our work demonstrates the potential of array-based single cell cytometry for kinetic analysis of signaling cascades in a high-throughput format.

Cell motility on polyethylene glycol block copolymers correlates to fibronectin surface adsorption.

Adhesion and motility of cells on polyethylene glycol (PEG) engineered surfaces are of fundamental interest for the development of biotechnological devices. Here, the structure of PEG block copolymers physisorbed to surfaces by polyLlysine (PLL) or polypropylene oxide (PPO) is studied. Cell behavior on such surfaces incubated with fibronectin (FN) is analyzed via time-lapse microscopy, the amount and the location of FN is determined via neutron reflectivity. While FN does not adsorb onto PPOPEG, 0.4-0.7 mg m(-2) of FN is found in the vicinity of the PLL moiety of PLLPEG. Cells exhibit 21% increased motility on PLLPEG (5 kDa PEG chains) compared to pure FN layers, and 12% decreased motility for PLLPEG (2 kDa PEG chains). These findings suggest that by design of PEGylated surfaces cell migration can be controlled.

Cellular self-organization on micro-structured surfaces.

Micro-patterned surfaces are frequently used in high-throughput single-cell studies, as they allow one to image isolated cells in defined geometries. Commonly, cells are seeded in excess onto the entire chip, and non-adherent cells are removed from the unpatterned sectors by rinsing. Here, we report on the phenomenon of cellular self-organization, which allows for autonomous positioning of cells on micro-patterned surfaces over time. We prepared substrates with a regular lattice of protein-coated adhesion sites surrounded by PLL-g-PEG passivated areas, and studied the time course of cell ordering. After seeding, cells randomly migrate over the passivated surface until they find and permanently attach to adhesion sites. Efficient cellular self-organization was observed for three commonly used cell lines (HuH7, A549, and MDA-MB-436), with occupancy levels typically reaching 40-60% after 3-5 h. The time required for sorting was found to increase with increasing distance between adhesion sites, and is well described by the time-to-capture in a random-search model. Our approach thus paves the way for automated filling of cell arrays, enabling high-throughput single-cell analysis of cell samples without losses.