Two-Bubble Acoustic Tweezing Cytometry for Biomechanical Probing and Stimulation of Cells

Abstract

The study of mechanotransduction relies on tools that are capable of applying mechanical forces to elicit and assess cellular responses. Here we report a new (to our knowledge) technique, called two-bubble acoustic tweezing cytometry (TB-ATC), for generating spatiotemporally controlled subcellular mechanical forces on live cells by acoustic actuation of paired microbubbles targeted to the cell adhesion receptor integrin. By measuring the ultrasound-induced activities of cell-bound microbubbles and the actin cytoskeleton contractile force responses, we determine that TB-ATC elicits mechanoresponsive cellular changes via cyclic, paired displacements of integrin-bound microbubbles driven by the attractive secondary acoustic radiation force (sARF) between the bubbles in an ultrasound field. We demonstrate the feasibility of dual-mode TB-ATC for both subcellular probing and mechanical stimulation. By exploiting the robust and unique interaction of ultrasound with microbubbles, TB-ATC provides distinct advantages for experimentation and quantification of applied forces and cellular responses for biomechanical probing and stimulation of cells.

Acoustic tweezing cytometry enhances osteogenesis of human mesenchymal stem cells through cytoskeletal contractility and YAP activation

“…Under the influence of US pulses, MBs underwent volume pulsation at the US frequency of 1 MHz, generating fluid microstreaming surrounding MBs that exert a local shear force to MB-loaded hMSCs [26]. More notably, during each US pulse (duration of 50 ms), the US field generated a directional acoustic radiation force of 30.62 nN on an individual MB, displacing the MB without detaching it from the cells, thereby generating local forces to MB-loaded hMSCs by straining the RGD-integrin-cytoskeleton connection [25, 26]. Between US pulses, the MBs retracted back toward their original positions on the cell membrane when there was no acoustic radiation force acting on the MBs.…”

“…In this work, we have leveraged a novel ultrasound-based method, the ATC, to utilize US pulses to actuate functionalized lipid MBs covalently attached to the cell surface of hMSCs to exert subcellular mechanical forces to the cells [25, 26]. US excitation of MBs elicits a rapid and sustained reactive cytoskeletal contractile force increase in mechanosensitive hMSCs [25, 26]. In this work, we have further extended the application of ATC and unequivocally demonstrated its capability for improving hMSC osteogenesis under culture conditions that are normally repressive for hMSC osteogenesis ( e.g.…”

“…We have recently developed a novel method, acoustic tweezing cytometry (ATC), for applying controllable subcellular forces to a large number of cells simultaneously [25, 26] (Fig. 1A).…”

“…Ultrasound (US) pulses are then applied to generate volume pulsation and translational movement of the integrin-anchored MBs, thereby exerting external forces in pN–nN range to the cells at the location of MB attachment. We have previously shown that ATC application can elicit a rapid and sustained increase in the cytoskeletal contractility of adherent mechanosensitive cells, including hMSCs [25, 26], demonstrating the potential of applying ATC for biomechanical stimulation of adherent mechanosensitive cells using MBs as an actuatable and biocompatible agent [25–27]. …”

Biophysical Tools for Cellular and Subcellular Mechanical Actuation of Cell Signaling

“…When two microbubbles are bound to a cell, the scattered acoustic fields from one microbubble generate a secondary acoustic radiation force on the neighboring microbubble. This can have a dramatic effect on microbubble behaviors in which microbubbles in an ultrasound field pulsating in phase are attracted to one another, regardless of the direction of the ultrasound wave (60). This type of mechanical actuation is quite unique and suitable for studying force-dependent adhesion-mediated signaling.…”