In vitro methods for evaluating therapeutic ultrasound exposures: present-day models and future innovations

Abstract

Although preclinical experiments are ultimately required to evaluate new therapeutic ultrasound exposures and devices prior to clinical trials, in vitro experiments can play an important role in the developmental process. A variety of in vitro methods have been developed, where each of these has demonstrated their utility for various test purposes. These include inert tissue-mimicking phantoms, which can incorporate thermocouples or cells and ex vivo tissue. Cell-based methods have also been used, both in monolayer and suspension. More biologically relevant platforms have also shown utility, such as blood clots and collagen gels. Each of these methods possesses characteristics that are well suited for various well-defined investigative goals. None, however, incorporate all the properties of real tissues, which include a 3D environment and live cells that may be maintained long-term post-treatment. This review is intended to provide an overview of the existing application-specific in vitro methods available to therapeutic ultrasound investigators, highlighting their advantages and limitations. Additional reporting is presented on the exciting and emerging field of 3D biological scaffolds, employing methods and materials adapted from tissue engineering. This type of platform holds much promise for achieving more representative conditions of those found in vivo, especially important for the newest sphere of therapeutic applications, based on molecular changes that may be generated in response to non-destructive exposures.

Keywords: Biological scaffolds; Ex vivo tissues; In vitro methods; Therapeutic ultrasound; Tissue-mimicking phantoms; Ultrasound bioeffects.

Figure 1

Optical and ultrasound visualization of different types of lesions in 6% BSA polyacrylamide TMM phantoms. The 'cigar’-shaped lesions (a) are typically created through thermal mechanisms only. The 'tadpole’-shaped (b) and 'egg’-shaped (c) lesions on the other hand are created by acoustic cavitation activity in the prefocal region (the FUS ultrasound transducer was on the right side). This interpretation is supported by the fact that the cavitation-based lesions are more visible by ultrasound due to the enhanced echogenicity of these regions (reprinted with permission from [11]).

Figure 2

Nanoparticle uptake in type I collagen gels. pFUS exposures in the gels were provided at a single location, after which the gels were immersed in a suspension of 100-nm diameter, fluorescently labeled, polystyrene NPs. In all three gels (a, b, and c), the NPs were initially taken up only in the region of treatment. Even at 24 h later, the NPs were somewhat more diffuse but still found to be restricted to the treated region (VF, unpublished).

Figure 3

Experimental setups for ultrasound treatment of cultured cells. (a) The ultrasound transducer (T) is positioned directly below a culture well containing the cells (S). Acoustic gel is used to couple between the transducer and the well. (b) Degassed water is used to couple between the transducer and the sample. An ultrasound absorber (UA) is used to prevent the reflection of the ultrasound waves. (c) Similar to setup B however with a variation in orientation. (d) The ultrasound transducer is inserted into the well. This setup is typically used for small samples in 24- or 96-well plates (reprinted with permission from [29]).

Figure 4

Experimental setup for investigating cavitation activity in agar gel tunnels. (left) A schematic representation of the setup showing the integration of the different elements that were used. (right) A photograph of the setup showing the gel, the FUS transducer, and the cavitation detector. All the components were in an acrylic tank filled with degassed water used for coupling. Microbubbles were injected into the tunnels just prior to the exposures (reprinted with permission from [36]).

Figure 5

Chitosan-gelatin biological scaffolds. (left) 2D scaffold: (a) brightfield image showing the fibrous structure of the scaffold; (b) fluorescent image of the same scaffold in (a), where the nuclei of fibroblasts are visible, stained with DAPI. Bar = 100 μm. (right) 3D scaffolds sectioned, stained with Masson's trichrome (red, scaffold; purple, fibroblasts), and observed with brightfield microscopy. (c) Edge region of a non-cellularized scaffold (bar = 200 μm). (inset) Entire scaffold (height = 7 mm; radius = 20 mm). (d,e) Regions of cellularized scaffolds (outer surface at top) (bar = 50 μm). Pore sizes range from 50 to 200 μm, with various degrees of cellularization.