High‐content imaging with micropatterned multiwell plates reveals influence of cell geometry and cytoskeleton on chromatin dynamics

Abstract

Understanding the mechanisms underpinning cellular responses to microenvironmental cues requires tight control not only of the complex milieu of soluble signaling factors, extracellular matrix (ECM) connections and cell-cell contacts within cell culture, but also of the biophysics of human cells. Advances in biomaterial fabrication technologies have recently facilitated detailed examination of cellular biophysics and revealed that constraints on cell geometry arising from the cellular microenvironment influence a wide variety of human cell behaviors. Here, we create an in vitro platform capable of precise and independent control of biochemical and biophysical microenvironmental cues by adapting microcontact printing technology into the format of standard six- to 96-well plates to create MicroContact Printed Well Plates (μCP Well Plates). Automated high-content imaging of human cells seeded on μCP Well Plates revealed tight, highly consistent control of single-cell geometry, cytoskeletal organization, and nuclear elongation. Detailed subcellular imaging of the actin cytoskeleton and chromatin within live human fibroblasts on μCP Well Plates was then used to describe a new relationship between cellular geometry and chromatin dynamics. In summary, the μCP Well Plate platform is an enabling high-content screening technology for human cell biology and cellular engineering efforts that seek to identify key biochemical and biophysical cues in the cellular microenvironment.

Complementary, Semiautomated Methods for Creating Multidimensional PEG-Based Biomaterials

“…All cell culture supplies were purchased from Thermo Fisher Scientific (Waltham, MA 24 medical-grade, double-sided adhesive to fit the well plate bottom, and it was attached to the plate by hand ensuring that there were no air bubbles present. Holes were cut in the adhesive from a custom template designed in Adobe Illustrator CC 2014 (Adobe Systems, San Jose, CA) with a laser cutter (Epilog Mini 18 Laser, Golden, CO).…”

Manufacturing Cell Therapies Using Engineered Biomaterials

“…Alternatively, ECM components such as collagen, decellularized matrices or cell-derived matrices can be utilized to form more ‘organic,’ albeit less defined, scaffolds. Photo-lithography, microcontact printing and other patterning techniques can be used to pattern single cell shapes or multiple cell aggregates [55,56]. Other sophisticated fabrication techniques such as block copolymer micelle nanolithography allow subcellular, nanoscale ligand patterning, and control over parameters such as ligand spacing, presentation and focal adhesion size [57].…”

“…Increased understanding of sophisticated biological processes and specific cell-ECM interactions enable engineering of a wide range of tailored matrix-mediated cellular perturbations. For instance, substrate-mediated epigenetic regulation of matrix properties can guide MSC differentiation [36] and secretion [65], as well as chromatin remodeling [56] and reprogramming efficiency of iPSCs [66]. iPSC differentiation and tissue or organoid formation may be enhanced through the use of biomaterials [67,68].…”

High content analysis platform for optimization of lipid mediated CRISPR-Cas9 delivery strategies in human cells

“…Our platform exploits image cytometry and high content image analysis (HCA) in combination with a customized microcontact printed cell substrate [34,35] to simultaneously monitor non-viral delivery and editing in human cells. We use established image analysis methods [36] and design-of-experiments (DOE) based statistical techniques [37,38] to screen existing liposomal delivery materials and to optimize delivery inputs to maximize gene-editing outcomes.…”