Ultrasound-Controlled Manipulation of Cells and Spheroids in 3D-Bioprinted Engineered Tumor Models

Introduction: 3D bioprinting is an important tool for recapitulating complex tissue architectures and biophysical parameters of the tumor microenvironment, with significant applications for studying cancer progression in controlled in-vitro models. Bioprinting techniques can be utilized to create geometries which mimic tumor vasculature or the ductal structures of the mammary gland[1]. Genetic manipulation of cells within bioprints is a key technique to model how changes in oncogene expression affect cancer progression in the 3D microenvironment. However, thick 3D scaffolds hinder diffusion of traditional transfection vectors, limiting spatial and temporal control of genetic manipulation. Focused ultrasound, in combination with ultrasound-responsive gene delivery microparticles, is a promising trigger for remote manipulation of cells within hydrogel constructs, due to its multi-centimeter penetration depth and ability to be tightly focused to small volumes in tissue[2]. The objective of this study is to design a 3D bioprinting technique that allows ultrasound-responsive particles to be printed in a cell or spheroid-seeded hydrogel bioink and activated by focused ultrasound for remote-controlled gene delivery to a specified region of the print. We hypothesize that successful 3D printing and subsequent activation of these particles will result in a pattern of gene delivery defined by the ultrasound focal zone, enabling noninvasive, spatiotemporally-controlled manipulation of cell behavior.

Materials and

Methods: A CellInk BioX6 extrusion 3D bioprinter was loaded with a solution containing an alginate bioink precursor pre-mixed with either HEK293T cells or MCF10A breast epithelial spheroids along with lipid-based ultrasound-responsive microparticles. The gene delivery microparticles were coupled with either green fluorescent protein (GFP) plasmid or a plasmid coding for HER2 oncogenic protein fused to a fluorescent reporter. Extrusion-based bioprinting was performed via conical needle using the Freeform Reversible Embedding of Suspended Hydrogels (FRESH) method[3] or coaxial bioprinting with a 16G OD, 20G ID coaxial needle. FRESH-printed constructs were printed into a dissolvable gelatin bead support slurry (LifeSupport®, FluidForm) with calcium chloride in the bath to promote crosslinking of the alginate. Coaxial-printed constructs utilized calcium chloride in the sheath compartment to promote crosslinking of the core alginate. At approximately 1 hr post-printing, constructs were exposed to focused ultrasound with the 1 mm3 focal volume of the ultrasound beam centered within the printed hydrogel construct. Printed constructs were imaged via brightfield and fluorescence microscopy to evaluate particle integrity and cell transfection at 48 hr post-ultrasound exposure. Viability was assessed via Hoechst/ethidium homodimer-1 or calcein-AM/ethidium homodimer-1 staining.

Results, Conclusions, and Discussions: Successful bioprinting of alginate hydrogel filaments containing cells and the lipid-based ultrasound-responsive microparticles was confirmed via microscopy. Printed filament diameters with and without microparticles were modulated by varying printing tip gauges (Fig. 1A-B). The microparticles were observed to remain intact after extrusion bioprinting, and the particles were well-dispersed throughout the filaments. In response to applied focused ultrasound, the bioprinted particles ruptured only within the area of ultrasound exposure (Fig. 1C,F), while leaving the remaining particles intact. At 48 hours post-ultrasound exposure, GFP-expressing HEK293T cells were observed in the ultrasound focal zone via fluorescence microscopy (Fig. 1C,F), and the number of transfected cells was modulated by varying the number of applied ultrasound pulses (Fig. 1 C-E). Bioprinted breast epithelial (MCF10A) spheroids showed high cell viability of over 85% at 48 hours, and over 70% at 10 days post-printing in multiple bioink formulations (Fig. 1G-H). In spheroid bioprints with gene delivery microparticles, 48 hours post-ultrasound exposure, cells and cell clusters within spheroids in the ultrasound focal zone were seen to express GFP transgene (Fig. 1I,J). Additionally, in prints with breast epithelial spheroids along with HER2-coupled ultrasound-responsive microparticles (Fig. 1K), ultrasound-activated HER2 overexpression was observed in spheroid cells within the ultrasound focal zone. These results show that the responsive nature of the particles is maintained through the printing process and demonstrate that the prints can be activated with focused ultrasound for localized gene delivery. We developed 3D extrusion bioprinting methods that allow for noninvasive focused ultrasound-controlled particle activation and localized gene delivery to bioprinted cells and spheroids. This provides the first demonstration of cell-seeded 3D-bioprinted structures designed for ultrasound-activatable gene delivery. Furthermore, this technique was used to transfect healthy spheroids from a specified region of a bioprinted construct to overexpress HER2 oncogene. This remote-controlled gene delivery platform enables user-defined gene expression within bioprinted tumor constructs and can be leveraged to investigate the behavior and signaling of transformed cells in the 3D context. Future work will utilize this platform to study how complex tissue microenvironments affect the progression from isolated transformed cells to aggressive cancer in 3D tissues.

Acknowledgements (Optional): [1] Nerger B and Nelson CM. Biomaterials. 2019; 198:135-145.
[2] Gelmi A and Schutt CE. Adv. Healthcare Mater. 2021; 10:2001125.
[3] Hinton TJ, et al. Science Advances. 2015; 1:e1500758.