Development of a 3D printed perfusable in vitro blood-brain barrier model for use as a scalable screening tool.

Despite recent technological advances in drug discovery, the success rate for neurotherapeutics remains alarmingly low compared to treatments for other areas of the body. One of the biggest challenges for delivering therapeutics to the central nervous system (CNS) is the presence of the blood-brain barrier (BBB). In vitro blood-brain barrier models with high predictability are essential to aid in designing parameters for new therapeutics, assess their ability to cross the BBB, and investigate therapeutic strategies that can be employed to enhance transport. Here, we demonstrate the development of a 3D printable hydrogel blood-brain barrier model that mimics the cellular composition and structure of the blood-brain barrier with human brain endothelial cells lining the surface, pericytes in direct contact with the endothelial cells on the abluminal side of the endothelium, and astrocytes in the surrounding printed bulk matrix. We introduce a simple, static printed hemi-cylinder model to determine design parameters such as media selection, co-culture ratios, and cell incorporation timing in a resource-conservative and high-throughput manner. Presence of cellular adhesion junction, VE-Cadherin, efflux transporters, P-glycoprotein (P-gp) and Breast cancer resistance protein (BCRP), and receptor-mediated transporters, Transferrin receptor (TfR) and low-density lipoprotein receptor-related protein 1 (LRP1) were confirmed via immunostaining demonstrating the ability of this model for screening in therapeutic strategies that rely on these transport systems. Design parameters determined in the hemi-cylinder model were translated to a more complex, perfusable vessel model to demonstrate its utility for determining barrier function and assessing permeability to model therapeutic compounds. This 3D-printed blood-brain barrier model represents one of the first uses of projection stereolithography to fabricate a perfusable blood-brain barrier model, enabling the patterning of complex vessel geometries and precise arrangement of cell populations. This model demonstrates potential as a new platform to investigate the delivery of neurotherapeutic compounds and drug delivery strategies through the blood-brain barrier, providing a useful in vitro screening tool in central nervous system drug discovery and development.

FRESH™ 3D bioprinted cardiac tissue, a bioengineered platform for in vitro pharmacology.

There is critical need for a predictive model of human cardiac physiology in drug development to assess compound effects on human tissues. In vitro two-dimensional monolayer cultures of cardiomyocytes provide biochemical and cellular readouts, and in vivo animal models provide information on systemic cardiovascular response. However, there remains a significant gap in these models due to their incomplete recapitulation of adult human cardiovascular physiology. Recent efforts in developing in vitro models from engineered heart tissues have demonstrated potential for bridging this gap using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in three-dimensional tissue structure. Here, we advance this paradigm by implementing FRESH™ 3D bioprinting to build human cardiac tissues in a medium throughput, well-plate format with controlled tissue architecture, tailored cellular composition, and native-like physiological function, specifically in its drug response. We combined hiPSC-CMs, endothelial cells, and fibroblasts in a cellular bioink and FRESH™ 3D bioprinted this mixture in the format of a thin tissue strip stabilized on a tissue fixture. We show that cardiac tissues could be fabricated directly in a 24-well plate format were composed of dense and highly aligned hiPSC-CMs at >600 million cells/mL and, within 14 days, demonstrated reproducible calcium transients and a fast conduction velocity of ∼16 cm/s. Interrogation of these cardiac tissues with the ß-adrenergic receptor agonist isoproterenol showed responses consistent with positive chronotropy and inotropy. Treatment with calcium channel blocker verapamil demonstrated responses expected of hiPSC-CM derived cardiac tissues. These results confirm that FRESH™ 3D bioprinted cardiac tissues represent an in vitro platform that provides data on human physiological response.

A 3D printable perfused hydrogel vascular model to assay ultrasound-induced permeability.

The development of an in vitro model to study vascular permeability is vital for clinical applications such as the targeted delivery of therapeutics. This work demonstrates the use of a perfusion-based 3D printable hydrogel vascular model as an assessment for endothelial permeability and its barrier function. Aside from providing a platform that more closely mimics the dynamic vascular conditions in vivo, this model enables the real-time observation of changes in the endothelial monolayer during the application of ultrasound to investigate the downstream effect of ultrasound-induced permeability. We show an increase in the apparent permeability coefficient of a fluorescently labeled tracer molecule after ultrasound treatment via a custom MATLAB algorithm, which implemented advanced features such as edge detection and a dynamic region of interest, thus supporting the use of ultrasound as a non-invasive method to enhance vascular permeability for targeted drug therapies. Notably, live-cell imaging with VE-cadherin-GFP HUVECs provides some of the first real-time acquisitions of the dynamics of endothelial cell-cell junctions under the application of ultrasound in a 3D perfusable model. This model demonstrates potential as a new scalable platform to investigate ultrasound-assisted delivery of therapeutics across a cellular barrier that more accurately mimics the physiologic matrix and fluid dynamics.