High throughput screening of live cells is a crucial technology that allows for the parallel functional evaluation of the influence of multiple factors on cell behavior and phenotype. In the last years due to the rapid expansion of bioinformatics and genomic tools, increasing throughput and decreasing screening costs became an essential milestone for research in this field. In current study we present a Droplet-Array (DA) Sandwich Technology add reagents at any time point and retrieve the cells after culturing; (h) compatibility with standard screening microscopes. In the current study we demonstrate that DA Sandwich Chip can be applied for performing drug screens and gene overexpression experiments with 3 commonly used adherent cell lines and therefore can be adopted for various cell-based screening applications.

Cell-based high content phenotypic screenings are widely used in fundamental research, pharmaceutical industry, and healthcare to simultaneously evaluate the effects of multiple compounds or gene over-expressions/knockdown on the phenotype of cells. Most screenings, especially in the industrial sector, are performed using microplate technology, which relies on high reagents and cell consumption as well as on expensive liquid handling robotics. Developing miniaturized screening platforms has been an important topic in the past decade. In this study we demonstrate the applicability of the Droplet-Microarray platform based on superhydrophobic-superhydrophilic patterning for cell-based high throughput screenings. We show the homogeneous seeding of cells and culturing of different adherent cell lines in individual droplets of different sizes. We demonstrate pipetting-free medium exchange enabling cell cultures in miniaturized droplet arrays for up to 5 days. We establish the method of reverse transfection and reverse drug screenings in individual nanoliter-size droplets by printing the transfection mixtures or drug molecules directly onto superhydrophilic spots prior to cell seeding.

We report a method to rapidly fabricate alginate hydrogel particles of specific sizes and shapes.Our method is based on the formation of arrays of droplets of pre-hydrogel solutions on superhydrophobic-hydrophilic patterns using the process of discontinuous dewetting, followed by their gelation via the parallel addition of CaCl 2 to the individual droplets via the sandwiching method. We demonstrate that viability of living cells incorporated within the hydrogel particles is higher during the long-term cultivation than in the case of cells cultured in the bulk threedimensional hydrogel matrix. Incorporation of magnetic particles into the free-standing hydrogel particles containing living cells enabled ease manipulation of the particles using an external magnetic field.

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.