Elastomeric sensor surfaces for high-throughput single-cell force cytometry

Abstract

All cell types generate mechanical forces in the contexts of their single-cell or tissue-level physiological roles. Since aberrant force-generating phenotypes directly lead to diseases, cellular force-generation mechanisms are high-value targets for new therapies. We report a scalable microtechnology to embed single-cell force sensors into elastomers that seamlessly integrates with the multi-well plate format to leverage laboratory automation workflows and achieves ~100-fold improvements in throughput for single-cell force measurements. We perform highly-parallelized time-course studies investigating airway biology and show that airway smooth muscle cells isolated from fatally asthmatic patients exhibit innately greater, and more rapid force generation in response to agonist than non-diseased cells. By also simultaneously tracing agonist-induced calcium flux and contractility in the same single cells, we reveal that calcium level is ultimately a poor quantitative predictor of cellular force generation. Finally, our flexible bio-functionalization approach uniquely enabled quantification of phagocytic forces in 1,000s of individual human macrophages and revealed that initiation of this force is a digital rather than a proportional response to the proper immunogen.

Neuronal PAS Domain 2 (Npas2)‐Deficient Fibroblasts Accelerate Skin Wound Healing and Dermal Collagen Reconstruction

“…During migration, fibroblasts adhere to the extracellular matrix (ECM) through integrin molecules and generate a single‐cell traction force (Style et al, ). We used a recently developed single‐cell contraction assay that required cell adhesion to the FLECS printed with fibronectin (Koziol‐White et al, ; Pushkarsky et al, ). The FLECS assay showed that mouse dermal fibroblasts increased the cell contraction behavior by Npas2 KO mutation (Fig.…”

Application of FRET Biosensors in Mechanobiology and Mechanopharmacological Screening

“…For example, the contractile force screening (CFS) platform was developed based upon straightforward measurement of cellular contractile force and is suitable for scaling to medium to high throughput ( Park et al, 2015 ). Besides, fluorescently labeled elastomeric contractible surfaces (FLECS) embedded in elastomers enables the single-cell force measurements in a high throughput manner ( Pushkarsky et al, 2018 ). Using micro- and nanotopography, a 96-well plate platform was developed to screen topographies and drug-topography combinations that have short- and long-term effects on T cell activation and proliferation ( Hu et al, 2016 ).…”

Technologies for the Directed Evolution of Cell Therapies

“…We recently developed an approach to quantify single-cell force generation massively in parallel based on micropatterned fluorescently labeled elastic protein patterns. 67 These patterns are stretched in a manner correlated to applied stress and can be analyzed quickly by automated fluorescence microscopy and image analysis algorithms. As an example of a cell type with therapeutic potential, we applied the platform to quantify the force generated by macrophages during phagocytosis of antibody-opsonized patterns.…”

“…HIgG elicited the most contractile response from the largest fraction of macrophages; however, outlier macrophages were observed for each condition with high force. Adapted with permission from Pushkarsky et al 67 of concept in which they characterized the cell-killing behavior of natural killer cells and an immortalized natural killer cell line (NK-92), but no sorting was performed. 81 Several technical challenges include controlling the ratios of cells within drops, and high-speed sorting of drops following cell killing to recover the optimal performing phenotype.…”