(M-505) Ultrasound-Controlled Manipulation of Cells and Spheroids in 3D-Bioprinted Tissue Constructs

Introduction:: 3D bioprinting has emerged as a valuable tool to recapitulate complex tissue architectures and biophysical parameters of the tumor microenvironment for development of in-vitro cancer models. Bioprinting techniques can be utilized to create geometries which mimic tumor vasculature or the ductal structures of the mammary gland¹. A platform that enables remote genetic manipulation of cells within bioprinted structures over time could allow for better modeling the complex dynamic signaling environment and localized oncogene overexpression associated with disease progression. 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 tissue². 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 22G conical needle using the Freeform Reversible Embedding of Suspended Hydrogels (FRESH) method³. Constructs were printed into a dissolvable gelatin bead support slurry (LifeSupport®, FluidForm) with calcium chloride in the bath to promote crosslinking of the 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 calcein-AM and ethidium homodimer staining.

Results, Conclusions, and Discussions:: Results and Discussion: Successful bioprinting of alginate hydrogel filaments containing cells and the lipid-based ultrasound-responsive microparticles was confirmed via microscopy. 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. 1A-B, 1D-E), 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). In breast epithelial spheroid bioprints, at 48 hours post-ultrasound exposure, cells and cell clusters within spheroids in the ultrasound focal zone were seen to overexpress GFP (Fig. 1F). Additionally, in prints with breast epithelial spheroids along with HER2-coupled ultrasound-responsive microparticles (Fig. 1G-H), ultrasound-activated HER2 overexpression was observed in spheroid cells within the ultrasound focal zone (Fig. 1I). Both the non-spheroid HEK293T cell and breast epithelial spheroid bioprints showed high cell viability of over 80% at 48 hours. 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.
Conclusions: A 3D bioprinting method was developed to extrusion print ultrasound-responsive microparticles within alginate bioinks, allowing for noninvasive focused ultrasound-controlled particle activation and localized gene delivery within both bioprinted single cells and spheroids. This provides the first demonstration of a cell-seeded 3D-bioprinted structure 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 site-specific gene expression within bioprinted tumor constructs and can be leveraged to investigate the behavior and signaling of transformed cells in the 3D context. As this technique enables localized oncogene overexpression of cells within complex tissue microenvironments, it can be further utilized to model the progression from a single transformed cell to aggressive cancer. Future work will involve the addition of endothelial cells and additional microenvironmental features to our multi-cellular bioprints to model intravasation and cancer progression.

Acknowledgements (Optional): : M.K.L. acknowledges support from Knight Cancer Early Detection Advanced Research Center & Kuni Foundation.

References (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.