Analytical Performance Evaluation of New DESI Enhancements for Targeted Drug Quantification in Tissue Sections.

Desorption/ionization (DI)-mass spectrometric (MS) methods offer considerable advantages of rapidity and low-sample input for the analysis of solid biological matrices such as tissue sections. The concept of desorption electrospray ionization (DESI) offers the possibility to ionize compounds from solid surfaces at atmospheric pressure, without the addition of organic compounds to initiate desorption. However, severe drawbacks from former DESI hardware stability made the development of assays for drug quantification difficult. In the present study, the potential of new prototype source setups (High Performance DESI Sprayer and Heated Transfer Line) for the development of drug quantification assays in tissue sections was evaluated. It was demonstrated that following dedicated optimization, new DESI XS enhancements present promising options regarding targeted quantitative analyses. As a model compound for these developments, ulixertinib, an inhibitor of extracellular signal-regulated kinase (ERK) 1 and 2 was used.

Droplet-Microarray: Miniaturized Platform for High-Throughput Screening of Antimicrobial Compounds.

Currently, there are no time-saving and cost-effective high-throughput screening methods for the evaluation of bacterial drug-resistance. In this study, a droplet microarray (DMA) system is established as a miniaturized platform for high-throughput screening of antibacterial compounds using the emerging, opportunistic human pathogen Pseudomonas aeruginosa (P. aeruginosa) as a target. Based on the differences in wettability of DMA slides, a rapid method for generating microarrays of nanoliter-sized droplets containing bacteria is developed. The bacterial growth in droplets is evaluated using fluorescence. The new method enables immediate screening with libraries of antibiotics. A novel simple colorimetric readout method compatible with the nanoliter size of the droplets is established. Furthermore, the drug-resistance of P. aeruginosa 49, a multi-resistant strain from an environmental isolate, is investigated. This study demonstrates the potential of the DMA platform for the rapid formation of microarrays of bacteria for high-throughput drug screening.

Facile One Step Formation and Screening of Tumor Spheroids Using Droplet-Microarray Platform.

Tumor spheroids or microtumors are important 3D in vitro tumor models that closely resemble a tumor's in vivo "microenvironment" compared to 2D cell culture. Microtumors are widely applied in the fields of fundamental cancer research, drug discovery, and precision medicine. In precision medicine tumor spheroids derived from patient tumor cells represent a promising system for drug sensitivity and resistance testing. Established and commonly used platforms for routine screenings of cell spheroids, based on microtiter plates of 96- and 384-well formats, require relatively large numbers of cells and compounds, and often lead to the formation of multiple spheroids per well. In this study, an application of the Droplet Microarray platform, based on hydrophilic-superhydrophobic patterning, in combination with the method of hanging droplet, is demonstrated for the formation of highly miniaturized single-spheroid-microarrays. Formation of spheroids from several commonly used cancer cell lines in 100 nL droplets starting with as few as 150 cells per spheroid within 24-48 h is demonstrated. Established methodology carries a potential to be adopted for routine workflows of high-throughput compound screening in 3D cancer spheroids or microtumors, which is crucial for the fields of fundamental cancer research, drug discovery, and precision medicine.

Droplet microarray: miniaturized platform for rapid formation and high-throughput screening of embryoid bodies.

Stem cells are influenced by various factors present in their in vivo microenvironment, such as interactions with neighboring cells, the extracellular matrix or soluble molecules. This demonstrates the high complexity of the in vivo microenvironment. Hence, many advances have been made in developing 3D screening models mimicking this complexity and the in vivo-like state in order to ensure more biomedically relevant investigations in drug discovery. In the field of stem cell research embryoid bodies are often used as relevant 3D systems. Embryoid bodies are embryonic stem cell aggregates that recapitulate the early embryonic development and that can differentiate into derivatives of the three germ layers. Embryoid bodies enable the investigation of processes underlying embryonic development, tissue generation and identification of drugs with developmental toxicity. The ability to perform high-throughput screenings using embryoid bodies could be extremely important to accelerate the progress in the field of stem cell research and embryonic development. To date, there are no simple methods to create high-density microarrays of embryoid bodies that further enable their high-throughput screening important for biomedical research. Here we demonstrate a new method that enables formation and high-throughput screening of embryoid bodies in arrays of defined, separated microdroplets. Using the superhydrophobic-hydrophilic micropattern of the droplet microarray, we demonstrate rapid and facile one-step formation of a dense array of multiple droplets containing homogeneous, single embryoid bodies. Thorough characterization of the influence of the initial cell number on embryoid body size, roundness and distribution was performed. We applied the embryoid body microarray to screen 774 FDA-approved compounds, identifying compounds with developmental toxicity such as mycophenolate mofetil or embryonic lethality such as eptifibatide. This work demonstrates the potential of the droplet microarray for the rapid formation of high-density microarrays of single embryoid bodies and their high-throughput drug screenings.

Fabrication of Hydrogel Particles of Defined Shapes Using Superhydrophobic-Hydrophilic Micropatterns.

High-throughput fabrication of freestanding hydrogel particles with defined geometry and size for 3D cell culture, cell screenings, and modular tissue engineering is reported. The method employs discontinuous dewetting using superhydrophobic-hydrophilic micropatterns.

Droplet-Array (DA) Sandwich Chip: A Versatile Platform for High-Throughput Cell Screening Based on Superhydrophobic-Superhydrophilic Micropatterning.

A droplet-array (DA) sandwich chip is a miniaturized platform for cell-based high-throughput screening. It is based on sandwiching of a glass slide with a preprinted library and a superhydrophobic-superhydrophilic pattern, which consists of thousands of simultaneously formed microdroplets containing cells. The DA sandwich chip allows for one-step cell seeding, simultaneous initiation of screening, and 1000 times less reagent consumption than a regular 96-well plate.