Ultra-high speed imaging of cell-microbubble interactions for bone repair

Abstract

Lipid-stabilised, ultrasound-activated microbubbles are used as contrast agents in medical ultrasound imaging. They are more recently being exploited as minimally invasive site-specific drug delivery vehicles in oncology and regenerative medicine. However, the megahertz frequencies required for microbubble activation make the study of the mechanisms by which they exert effects on cells and tissue challenging. The aim of this study was to develop an acoustic device to image and quantify bone cell-microbubble interactions at sub-microsecond temporal resolution. A compact cell culture device for ultrasound stimulation and imaging was designed and built using PDMS moulding. MG-63 osteosarcoma cells were tested for viability in the device with calcein AM. Microbubbles were fabricated by sonication and attached to cells via flask inversion. 1 MHz ultrasound at 220 kPa was used to stimulate the culture. Ultra-high speed imaging was performed at x80 magnification with an Olympus IX71 microscope and Shimadzu HPV-X camera at 5 million frames per second. Digital image correlation (DIC) was performed using MATCH ID software to quantify cell deformations with respect to time, microbubble size, formulation and other parameters. Cells remained viable within the device for up to 72 hours and microbubbles adhered to them. UHS images of interactions were captured with sufficient temporal and spatial resolution to detect cell deformations and to perform DIC on images of single cells. Microbubble-induced cell deformations fitted to sinusoidal models and could be attributed to 1 MHz microbubble oscillations. Cell cytoplasm strain decayed as a function of distance from the microbubble, with a good fit to logarithmic or inverse square models. Cells that were stiffened by treatment with paraformaldehyde fixation had a faster deformation decay compared to those that did not. These data indicate that mechanical interactions between cells and microbubbles can be quantified with sub-microsecond resolution. This offers the opportunity to relate the physical effects of microbubble interaction to cell behaviour and provides a novel method to recover mechanical information about cells and tissues at megahertz strain rates.

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Identifiers

ORCID for Fabrice Pierron: orcid.org/0000-0003-2813-4994

ORCID for Nick Evans: orcid.org/0000-0002-3255-4388

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