Early screening of drug candidates for genotoxicity typically includes an analysis for mutagenicity in bacteria and for clastogenicity in cultured mammalian cells. In addition, in recent years, an early assessment of photogenotoxicity potential has become increasingly important. Also, for screening purposes, expert computer systems can be used to identify structural alerts. In cases where structural alerts are identified, mutagenicity testing limited to bacteria can be conducted. The sequence of computer-aided analysis and limited testing using bacteria allows for screening a comparatively large number of drug candidates. In contrast, considerably more resources, in terms of supplies, technical time, and the amount of a test substance needed, are required when screening for clastogenic activity in mammalian cells. In addition, the relatively large percentage of false positive results for rodent carcinogenicity associated with clastogenicity assays is of considerable concern. As a consequence, mammalian cell-based alternatives to clastogenicity assays are needed for early screening of mammalian genotoxicity. The comet assay is a relatively fast, simple, and sensitive technique for the analysis of DNA damage in mammalian cells. This assay seems especially useful for screening purposes because false positives associated with excessive toxicity appear to occur less frequently, only relatively small amounts of a test compound are needed, and certain steps of the test procedure can be automated. Therefore, the in vitro comet assay is proposed as an alternative to cytogenetic assays in early genotoxicity/photogenotoxicity screening of drug candidates.

Controlling the interfaces between cells and solid substrates is an important theme pertinent to a variety of research applications such as cell biology, [1] tissue engineering, [2] and cell-based sensors and chip devices. [3] In particular, the recent progress in the understanding of the molecular mechanism of cell adhesion has promoted the development of self-assembled monolayer (SAM)-based techniques for precise cell pattering, for example, microcontact printing (mCP).[4] SAMs that are terminated in short oligomers of the ethylene glycol (OEG) unit (HS(CH 2 ) 11 (OCH 2 CH 2 ) n OH; n = 3-7) were most often used in these studies for resisting the nonspecific adsorption of proteins/cells, and are proven to be the best "nonadsorption" systems that are currently available.[5] However, conventional mCP methods produce static surface stimuli. Once cells are adherent, it is difficult to subsequently alter the matrix environment.[6]Herein, we described a strategy for the real-time local manipulation of the cell-adhesive property of an OEGterminated SAM substrate using ultramicroelectrodes (UME), which enables the template-free formation of cellular micropatterns by directing cell adhesion and growth in situ. The strategy is based on our finding that the cytophobic nature of OEG SAMs is rapidly switched to cell adhesive by exposure to some oxidizing agents, such as Br 2 , which can be electrogenerated from Br À in aqueous solution. By scanning a UME as a "pen" closely above the substrate, one can draw a cellular pattern. As illustrated in Figure 1, an OEG-terminated SAM substrate was formed by inserting a gold slide into an OEG-terminated alkanethiol solution for 12 h. Then, a microelectrode with a radius r T = 25 mm (T = tip, of the UME) was brought to 5 mm above the substrate (Figure 1 a). A 5 s potential pulse of 1.2 V (compared with an Ag quasireference electrode) was applied in a 0.1m phosphate buffer solution containing 25 mm KBr (pH 7.4). The electrochemically generated Br 2 diffused to the substrate and quickly reacted with the OEG monolayer locally. The modification of the OEG SAM was confirmed by a scanning electrochemical microscopy (SECM) feedback image (Figure 1 b). The contrast is based on the permeability difference of the OEG SAM and the Br 2 -treated SAM. The bright parts in the image indicate higher reduction currents, corresponding to a higher permeability of the monolayer (see the Supporting Information). The treated substrate was then incubated in extracellular matrix (ECM) protein solutions, for example, fibronectin, fibrinogen etc. Proteins adsorbed exclusively on the electrochemically treated regions (Figure 1 c). Figure 1 d shows a confocal laser scanning fluorescent micrograph of ECM protein pattern formed after adsorption of a fluorescence-labeled protein (4 h in 100 mg mL À1 fibronogen-Alexa 488). During a subsequent seeding of human fibroblast culture on the substrate, the preadsorbed protein promoted the specific attachment of fibroblasts, resulting in the formation of a cellular micropat...

No abstract

Patterned cell cultures obtained by microcontact printing have been modified in situ by a microelectrochemical technique. It relies on lifting cell-repellent properties of oligo(ethylene glycol)-terminated self-assembled monolayers (SAMs) by Br2, which is produced locally by an ultramicroelectrode of a scanning electrochemical microscope (SECM). After Br2 treatment the SAM shows increased permeability and terminal hydrophobicity as characterized by SECM approach curves and contact angle measurements, respectively. Polarization-modulation Fourier transform infrared reflection-absorption spectroscopic (PM FTIRRAS) studies on macroscopic samples show that the Br2 treatment removes the oligo(ethelyene glycol) part of the monolayer within a second time scale while the alkyl part of the SAM degrades with a much slower rate. The lateral extension of the modification can be limited because heterogeneous electron transfer from the gold support destroys part of the electrogenerated Br2 once the monolayer is locally damaged in a SECM feedback configuration. This effect has been reproduced and analyzed by exposing SAM-modified samples to Br2 in the galvanic cell Au|SAM|5 microM Br2 + 0.1 M Na2SO4||10 microM KBr + 0.1 M Na2SO4|Au followed by an PM FTIRRAS characterization of the changes in the monolayer system.

Since the introduction of chemotherapy for cancer treatment in the early 20th century considerable efforts have been made to maximize drug efficiency and at the same time minimize side effects. As there is a great interpatient variability in response to chemotherapy, the development of predictive biomarkers is an ambitious aim for the rapidly growing research area of personalized molecular medicine. The individual prediction of response will improve treatment and thus increase survival and life quality of patients. In the past, cell cultures were used as in vitro models to predict in vivo response to chemotherapy. Several in vitro chemosensitivity assays served as tools to measure miscellaneous endpoints such as DNA damage, apoptosis and cytotoxicity or growth inhibition. Twenty years ago, the development of high-throughput technologies, e.g. cDNA microarrays enabled a more detailed analysis of drug responses. Thousands of genes were screened and expression levels were correlated to drug responses. In addition, mutation analysis became more and more important for the prediction of therapeutic success. Today, as research enters the area of -omics technologies, identification of signaling pathways is a tool to understand molecular mechanism underlying drug resistance. Combining new tissue models, e.g. 3D organoid cultures with modern technologies for biomarker discovery will offer new opportunities to identify new drug targets and in parallel predict individual responses to anticancer therapy. In this review, we present different currently used chemosensitivity assays including 2D and 3D cell culture models and several –omics approaches for the discovery of predictive biomarkers. Furthermore, we discuss the potential of these assays and biomarkers to predict the clinical outcome of individual patients and future perspectives.