High-throughput single-cell assay for precise measurement of the intrinsic mechanical properties and shape characteristics of red blood cells.

The intrinsic physical and mechanical properties of red blood cells (RBCs), including their geometric and rheological characteristics, can undergo changes in various circulatory and metabolic diseases. However, clinical diagnosis using RBC biophysical phenotypes remains impractical due to the unique biconcave shape, remarkable deformability, and high heterogeneity within different subpopulations. Here, we combine the hydrodynamic mechanisms of fluid-cell interactions in micro circular tubes with a machine learning method to develop a relatively high-throughput microfluidic technology that can accurately measure the shear modulus of the membrane, viscosity, surface area, and volume of individual RBCs. The present method can detect the subtle changes of mechanical properties in various RBC components at continuum scales in response to different doses of cytoskeletal drugs. We also investigate the correlation between glycosylated hemoglobin and RBC mechanical properties. Our study develops a methodology that combines microfluidic technology and machine learning to explore the material properties of cells based on fluid-cell interactions. This approach holds promise in offering novel label-free single-cell-assay-based biophysical markers for RBCs, thereby enhancing the potential for more robust disease diagnosis.

Mechanosensitive accumulation of non-muscle myosin IIB during mitosis requires its translocation activity.

Non-muscle myosin II (NMII) is a force-generating mechanosensitive enzyme that responds to mechanical forces. NMIIs mechanoaccumulate at the cell cortex in response to mechanical forces. It is essential for cells to mechanically adapt to the physical environment, failure of which results in mitotic defects when dividing in confined environment. Much less is known about how NMII mechanoaccumulation is regulated during mitosis. We show that mitotic cells respond to compressive stress by promoting accumulation of active RhoA at the cell cortex as in interphase cells. RhoA mechanoresponse during mitosis activates and stabilizes NMIIB via ROCK signaling, leading to NMIIB mechanoaccumulation at the cell cortex. Using disease-related myosin II mutations, we found that NMIIB mechanoaccumulation requires its motor activity that translocates actin filaments, but not just its actin-binding function. Thus, the motor activity coordinates structural movement and nucleotide state changes to fine-tune actin-binding affinity optimal for NMIIs to generate and respond to forces.

Evolution of surface area and membrane shear modulus of matured human red blood cells during mechanical fatigue.

Mechanical properties of red blood cells (RBCs) change during their senescence which supports numerous physiological or pathological processes in circulatory systems by providing crucial cellular mechanical environments of hemodynamics. However, quantitative studies on the aging and variations of RBC properties are largely lacking. Herein, we investigate morphological changes, softening or stiffening of single RBCs during aging using an in vitro mechanical fatigue model. Using a microfluidic system with microtubes, RBCs are repeatedly subjected to stretch and relaxation as they squeeze into and out of a sudden contraction region. Geometric parameters and mechanical properties of healthy human RBCs are characterized systematically upon each mechanical loading cycle. Our experimental results identify three typical shape transformations of RBCs during mechanical fatigue, which are all strongly associated with the loss of surface area. We constructed mathematical models for the evolution of surface area and membrane shear modulus of single RBCs during mechanical fatigue, and quantitatively developed an ensemble parameter to evaluate the aging status of RBCs. This study provides not only a novel in vitro fatigue model for investigating the mechanical behavior of RBCs, but also an index closely related to the age and inherent physical properties for a quantitative differentiation of individual RBCs.