Christina Rommel

Cytotoxicity screening of 23 engineered nanomaterials using a test matrix of ten cell lines and three different assays

BackgroundEngineered nanomaterials display unique properties that may have impact on human health, and thus require a reliable evaluation of their potential toxicity. Here, we performed a standardized in vitro screening of 23 engineered nanomaterials. We thoroughly characterized the physicochemical properties of the nanomaterials and adapted three classical in vitro toxicity assays to eliminate nanomaterial interference. Nanomaterial toxicity was assessed in ten representative cell lines.ResultsSix nanomaterials induced oxidative cell stress while only a single nanomaterial reduced cellular metabolic activity and none of the particles affected cell viability. Results from heterogeneous and chemically identical particles suggested that surface chemistry, surface coating and chemical composition are likely determinants of nanomaterial toxicity. Individual cell lines differed significantly in their response, dependent on the particle type and the toxicity endpoint measured.ConclusionIn vitro toxicity of the analyzed engineered nanomaterials cannot be attributed to a defined physicochemical property. Therefore, the accurate identification of nanomaterial cytotoxicity requires a matrix based on a set of sensitive cell lines and in vitro assays measuring different cytotoxicity endpoints.

Simplified approach for quantitative digital holographic phase contrast imaging of living cells

Many interferometry-based quantitative phase contrast imaging techniques require a separately generated coherent reference wave. This results in a low phase stability and the demand for a precise adjustment of the intensity ratio between object and reference wave. To overcome these problems, the performance of a Michelson interferometer approach for digital holographic microscopy was analyzed that avoids a separately generated reference wave by superposition of different image areas. It is shown that this simplified arrangement yields improved phase stability. Furthermore, results from time-lapse investigations on living pancreas tumor cells demonstrate the capability of the method for reliable quantitative phase contrast imaging.

Contrast-enhanced digital holographic imaging of cellular structures by manipulating the intracellular refractive index

The understanding of biological reactions and evaluation of the significance for living cells strongly depends on the ability to visualize and quantify these processes. Digital holographic microscopy (DHM) enables quantitative phase contrast imaging for high resolution and minimal invasive live cell analysis without the need of labeling or complex sample preparation. However, due to the rather homogeneous intracellular refractive index, the phase contrast of subcellular structures is limited and often low. We analyze the impact of the specific manipulation of the intracellular refractive index by microinjection on the DHM phase contrast. Glycerol is chosen as osmolyte, which combines high solubility in aqueous solutions and biological compatibility. We show that the intracellular injection of glycerol causes a contrast enhancement that can be explained by a decrease of the cytosolic refractive index due to a water influx. The underlying principle is proven by experiments inducing cell shrinkage and with fixated cells. The integrity of the cell membrane is considered as a prerequisite and allows a reversible cell swelling and shrinking within a certain limit. The presented approach to control the intracellular phase contrast demonstrated for the example of DHM opens prospects for applications with other quantitative phase contrast imaging methods.

Elasticity Mapping of Pore‐Suspending Native Cell Membranes

The mechanics of cellular membranes are governed by a non-equilibrium composite framework consisting of the semiflexible filamentous cytoskeleton and extracellular matrix proteins linked to the lipid bilayer. While elasticity information of plasma membranes has mainly been obtained from whole cell analysis, techniques that allow addressing local mechanical properties of cell membranes are desirable to learn how their lipid and protein composition is reflected in the elastic behavior on local length scales. Introduced here is an approach based on basolateral membranes of polar epithelial Madin-Darby canine kidney (MDCK) II cells, prepared on a highly ordered porous substrate that allows elastic mapping on a submicrometer-length scale. A strong correlation between the density of actin filaments and the measured membrane elasticity is found. Spatially resolved indentation experiments carried out with atomic force and fluorescence microscope permit relation of the supramolecular structure to the elasticity of cellular membranes. It is shown that the elastic response of the pore spanning cell membranes is governed by local bending modules rather than lateral tension.

Cell membrane topology analysis by RICM enables marker-free adhesion strength quantification

Reflection interference contrast microscopy (RICM) allows the visualization of the cell’s adhesion topology on substrates. Here it is applied as a new label-free method to measure adhesion forces between tumor cells and their substrate without any external manipulation, i.e., the application of force or adjustments in the substrate elasticity. Malignant cancer transformation is closely associated with the down-regulation of adhesion proteins and the consequent reduction of adhesion forces. By analyzing the size and distribution of adhesion patches from a benign and a malignant human pancreatic tumor cell line, we established a model for calculating the adhesion strength based on RICM images. Further, we could show that the cell’s spread area does not necessarily scale with adhesion strength. Despite the larger projected cell area of the malignant cell line, adhesion strength was clearly reduced. This underscores the importance of adhesion patch analysis. The calculated force values were verified by microfluidic detachment assays. Static and dynamic RICM measurements produce numerous adhesion-related parameters from which characteristic cell signatures can be derived. Such a cellular fingerprint can refine the process of categorizing cell lines according to their grade of differentiation.

Elasticity mapping of apical cell membranes

Apical cell membranes obtained from polar epithelial MDCK II cells were prepared on a highly ordered porous substrate, which allows local elastic mapping by force indentation curves.

Multimodal label-free in vitro toxicity testing with digital holographic microscopy

Common in vitro toxicity tests of drugs, chemicals or nanomaterials involves the measurement of cellular endpoints like stress response, cell viability, proliferation or cell death. The assay systems determine enzyme activity or protein expression by optical read out of enzyme substrates or marker protein labeling. These standard procedures have several disadvantages. Cellular processes have to be stopped at a distinct time point for the read out, where usually only parts of the cells were affected by the treatment with substances. Typically, only one parameter is analyzed and detection of cellular processes requires several time consuming incubations and washing steps. Here we have applied digital holographic microscopy (DHM) for a multimodal label-free analysis of drug toxicity. NIH 3T3 cells were incubated with 1 μM Taxol for 24 h. The recorded quantitative phase images were analyzed for cell thickness, cell volume, dry mass and cell migration. Taxol treated cells showed rapidly decreasing cell motility as measure of cell viability. A short increase in cell thickness and dry mass indicated cell division and growth in control cells, whereas Taxol treatment resulted in a continuous increase in cell height followed by a rapid decrease and a decrease of dry mass as indicators of cell death. Multimodal DHM analysis of drug treatment by multiple parameters allows direct and label-free detection of several toxicity parameters in parallel. DHM can quantify cellular reactions minimally invasive over a long time period and analyze kinetics of delayed cellular responses. Our results demonstrate digital holographic microscopy as a valuable tool for multimodal toxicity testing.

Macroporous silicon chips for laterally resolved, multi-parametric analysis of epithelial barrier function

This study describes a novel assay to visualize the macromolecular permeability of epithelial and endothelial cell layers with subcellular lateral resolution. Defects within the cell layer and details about the permeation route of the migrating solute are revealed. The assay is based on silicon chips with densely packed, highly ordered, dead-ended pores of μm-diameters on one side. The cells under study are grown on the porous side of the chip such that the pores in the growth surface serve as an array of femtolitre-sized cuvettes in which the permeating probe accumulates at the site of permeation. The pattern of pore filling reveals the permeability characteristics of the cell layer with a lateral resolution in the μm range. Coating of the chip surface with a thin layer of gold allows for impedance analysis of the adherent cells in order to measure their tightness for inorganic ions at the same time. The new assay provides an unprecedented look on epithelial and endothelial barrier function.

Standardized cell samples for midIR technology development

The application of midIR spectroscopy towards human cell and tissue samples is impaired by the need for technical solutions and lacking sample standards for technology development. We here present the standardization of stable test samples for the continuous development and testing of novel optical system components. We have selected cell lines representing the major cellular skin constituents keratinocytes and fibroblasts (NIH-3T3, HaCaT). In addition, two skin cancer cell types (A-375 and SK-MEL-28 cells) were analyzed. Cells were seeded on CaF2 substrates and measured dried and under aqueous medium as well as fixated and unfixated. Several independent cell preparations were analyzed with an FTIR spectrometer in the wave number range from 1000 - 4000 cm-1. The obtained data demonstrate that fixed and dehydrated cells on CaF2 can be stored in pure ethanol for several weeks without significant losses in quality of the spectral properties. The established protocol of cell seeding on CaF2 substrates, chemical fixation, dehydration, storage under ethanol and air-drying is suitable for the production of reliable midIR standards. The retrieved spectra demonstrate that fixed cells on CaF2 can be prepared reproducibly; with stable midIR spectral properties over several weeks and properties mimicking reliable unfixed cells. Moreover, the fixated samples on CaF2 show clear differences in the cell type specific spectra that can be identified by principle component analysis. In summary, the standardized cell culture samples on CaF2 substrates are suitable for the development of a midIR device and the optimization of the specific midIR spectra.

Multimodal optical phenotyping of cancer cells

There is a growing interest in label-free, optical techniques like digital holographic microscopy (DHM) and optical cell stretching, since the interaction with samples is minimized. Because optical manipulation strongly depends on the optical and physiological properties of the investigated material, we combined the usage of these methods for the characterization of pancreatic tumor cells. Our results demonstrate that cells of distinct differentiation levels, or different expression in only one protein, show differences in their deformability. Additionally, the DHM results showed only few variations in the refractive index, indicating that it does not significantly influence the results of the optical cell stretching. Thus, the combined usage of the two technologies represents a promising new approach for tumor cell characterization.