A High‐Throughput Automated Confocal Microscopy Platform for Quantitative Phenotyping of Nanoparticle Uptake and Transport in Spheroids

There is a high demand for advanced, image‐based, automated high‐content screening (HCS) approaches to facilitate phenotypic screening in 3D cell culture models. A major challenge lies in retaining the resolution of fine cellular detail but at the same time imaging multicellular structures at a large scale. In this study, a confocal microscopy‐based HCS platform in optical multiwell plates that enables the quantitative morphological profiling of populations of nonuniform spheroids obtained from HT‐29 human colorectal cancer cells is described. This platform is then utilized to demonstrate a quantitative dissection of the penetration of synthetic nanoparticles (NP) in multicellular 3D spheroids at multiple levels of scale. A pilot RNA interference‐based screening validates this methodology and identifies a subset of RAB GTPases that regulate NP trafficking in these spheroids. This technology is suitable for high‐content phenotyping in 3D cell‐based screening, providing a framework for nanomedicine drug development as applied to translational oncology.     

Automated Buildup of Biomimetic Films in Cell Culture Microplates for High‐Throughput Screening of Cellular Behaviors

An automatic method is established for layer‐by‐layer (LbL) assembly of biomimetic coatings in cell culture microplates using a commercial liquid‐handling robot. Highly homogeneous thin films are formed at the bottom of each microwell. The LbL film‐coated microplates are compatible with common cellular assays, using microplate readers and automated microscopes. Cellular adhesion is screened on crosslinked and peptide‐functionalized LbL films and stem cell differentiation in response to increasing doses of bone morphogenetic proteins (2, 4, 7, 9). This method paves the way for future applications of LbL films in cell‐based assays for regenerative medicine and high‐throughput drug screening.     

Inertial Microfluidic Cell Stretcher (iMCS): Fully Automated,High‐Throughput,and Near Real‐Time Cell Mechanotyping

Mechanical biomarkers associated with cytoskeletal structures have been reported as powerful label‐free cell state identifiers. In order to measure cell mechanical properties, traditional biophysical (e.g., atomic force microscopy, micropipette aspiration, optical stretchers) and microfluidic approaches were mainly employed; however, they critically suffer from low‐throughput, low‐sensitivity, and/or time‐consuming and labor‐intensive processes, not allowing techniques to be practically used for cell biology research applications. Here, a novel inertial microfluidic cell stretcher (iMCS) capable of characterizing large populations of single‐cell deformability near real‐time is presented. The platform inertially controls cell positions in microchannels and deforms cells upon collision at a T‐junction with large strain. The cell elongation motions are recorded, and thousands of cell deformability information is visualized near real‐time similar to traditional flow cytometry. With a full automation, the entire cell mechanotyping process runs without any human intervention, realizing a user friendly and robust operation. Through iMCS, distinct cell stiffness changes in breast cancer progression and epithelial mesenchymal transition are reported, and the use of the platform for rapid cancer drug discovery is shown as well. The platform returns large populations of single‐cell quantitative mechanical properties (e.g., shear modulus) on‐the‐fly with high statistical significances, enabling actual usages in clinical and biophysical studies.     

Nanoparticle patterning: High‐Resolution,Parallel Patterning of Nanoparticles via an Ion‐Induced Focusing Mask (Small 19/2010)

An ion‐induced focusing mask under the simultaneous injection of ions and charged aerosols generates invisible electrostatic lenses around each opening, through which charged nanoparticles are convergently guided without depositing on the mask surface. The sizes of the created features become significantly smaller than those of the mask openings due to the focusing capability. It is not only demonstrated that material‐independent nanoparticles including proteins can be patterned as an ordered array on any surface regardless of the conductive, nonconductive, or flexible nature of the substrate, but also that the array density can be increased. Highly sensitive gas sensors based on these focused nanoparticle patterns are fabricated via the concept.