Ultrasound Tweezers Trigger Single-Cell Mechanical Dynamics for Cell Mechanophenotyping

Cells actively modulate mechanobiological circuitry against external perturbations to stabilize whole cell/tissue physiology.The dynamic adaption of cells to mechanical force is critical for cells to perform vital biological functions,from single cell migration to embryonic development.Dysregulation of such dynamics has been associated with pathophysiological conditions in cardiovascular diseases,cancer,aging,and developmental disorders[1].Therefore,a direct understanding of cell’s biomechanical adaptive/maladaptive behaviors and the trigger factors causing the transformation of healthy adaption to maladaptation can help reveal the regulatory role of single cell mechanosensitive dynamics in the progression of various degenerative diseases and aging.However,current efforts for uncovering fundamental associations between disease and cell architecture have been focusing on'static'measurements of biophysical properties,which is limited by the requirement of large sample sizes to obtain statistically significant data.We therefore developed a single and highly integrated platform with mechanical stimulation and fine spatiotemporal sensing functions to probe the single cell mechanical dynamics at subcellular level to determine cell’s mechanophenotypes in healthy and disease conditions.We developed an integrated micromechanical system composed of an’ultrasound tweezer’stimulator[2]and a PDMS micropillar array [3] cellular force sensor to in situ noninvasively probe and monitor single cell mechanical dynamics.Vascular smooth muscle cells(VSMCs)from healthy mouse and mouse with induced abdominal aorta aneurysm(AAA)were used for cell mechanobiological study.An ultrasound transducer(V312-SM,Olympus)was used to generate ultrasound pulses to excite lipid-encapsulated microbubbles(Targeson)binding to cell membrane through an RGD-integrin linkage to apply a transient nanonewton force to VSMCs seeded on the PDMS micropillar array.PDMS micropillar array was fabricated and functionalized as previously described [3] and acts as the mechanical force sensor in our platform.Upon a 1 HZ and 10-second ultrasound stimulation,calcium influx was clearly detected in both healthy and AAA-VSMCs by using the fluo-4 calcium sensor,suggesting the microbubble-integrin-actin cytoskeleton(CSK)linkage can serve as a mechanosensory to sense the ultrasound stimulation.We then examined how healthy and AAA VSMCs would exhibit adaptions to mechanical stimulation at a global cellular scale.After the onset of a 10-second ultrasound stimulation,control and AAA-VSMCs displayed distinct dynamics of CSK tension within 30 mins,in which the CSK tension of healthy VSMCs increased within the reinforcement period(0-5 min)and restored to their ground state with the relaxation period(5-10 min);yet AAA-VSMCs displayed compromised dynamics of such CSK tension upon calcium influx.Quantitative analysis and theoretical modelling revealed the critical roles of myosin motor contraction,F-actin filament polymerization in regulating cell mechanosensitive dynamics in response to a transient mechanical perturbation.The distinct force and CSK dynamics in healthy and AAA conditions indicates that the force-dependent CSK molecular kinetics is a critical factor governing the distinct mechanosensitive dynamics of cells under pathologically dysfunctional conditions.Our results reveal that the mechanical adaptive process of cells to mechanical stimulus can measure the cellular mechanobiological phenotypes featured in both pathologically healthy and diseased context.We demonstrated that an altered mechanobiological phenotype,i.e.AAA-VSMCs with distinct actomyosin-CSK properties potentiates a mechanical maladaptation that reflects progressive accumulation of cellular damage and dysfunction.This may further reveal the pathogenic contexts and their physical mediators featuring biophysical dysregulation in cardiovascular diseases.