Embodiments of additive manufacturing that utilize hydrogels as building materials have recently received much attention for their ability to construct biological systems in vitro. These embodiments, often referred to as 3D bioprinters, build up layers of cell-laden hydrogel by rasterizing two dimensional patterns of material deposited in a layer-by-layer fashion; the most common mechanisms of pattern deposition include extrusion, where a shear-thinning gel is forced through a thin nozzle, and light-induced polymerization, where a laser polymerizes material from a vat of liquid hydrogel precursor solution. These methods of material deposition work well for hydrogels which are designed and optimized for their respective deposition method, however, many unique and useful designer hydrogels cannot be printed using conventional 3D bioprinters. Herein, we describe a novel method for layer-by-layer fabrication of hydrogel structures using open microfluidic patterning. For each layer of a printed hydrogel structure, a hydrophilic track or “rail” is manually placed parallel to and several hundred micrometers above the previously patterned layer. Hydrogel precursor solution is then introduced between the underlying layer and the rail, and flows along the rail via capillary action. The cross-sectional geometry of the rail constrains fluid flow to the space directly under the rail, meaning that the pattern of each layer of hydrogel is defined by the pattern of the rail corresponding to that layer. We show that this methodology can be used to fabricate relatively large (1 cm^3) structures of agarose gel, as well as cell laden structures of collagen and a novel peptide-based synthetic hydrogel. Finally, we show that the patterning rail can constrain fluid flow via differential surface chemistry, opening up the possibility for an automated 3D printer and advancing the commercial viability of the method.

Though renewed efforts in tuberculosis (TB) research have facilitated massive strides in treating Mycobacterium tuberculosis (Mtb), TB remains a global health problem with an estimated 10 million infections and 1.5 million deaths in 2018. The ability of the pathogen to sequester itself inside a granuloma, a mass of immune cells whose precise mechanism of regulation is unknown, prevents the simple study of Mtb pathogenesis and subsequent treatment discovery. Current in vivo models have been established to study TB infection using animal models or tissues, limiting biological relevance of human disease while current in vitro models lack components of the complex lung microenvironment during infection. We present the creation of a novel microscale infection model, which uses open and suspended microfluidic principles to enable spatial and temporal manipulation of cultures in suspended hydrogel plugs. Utilizing the ‘stacking’ feature of the device, we demonstrate the ability of a model granuloma consisting of M.bovis BCG (Mycobacterium bovis bacille Calmette-Guérin) and monocyte-derived macrophages to interact with a model vasculature layer consisting of endothelial cells. Analysis of soluble factors for proinflammatory cytokines and characterization of infection-dependent angiogenesis in the vasculature layer are used to verify crosstalk between cultures. In the future, we envision this model expanding to contain multiple immune cell types and to incorporate additional aspects of the lung anatomy to approach a more accurate pathophysiological model as a tool for other researchers’ studies.

There are an estimated 300 million people worldwide who are affected by asthma, a respiratory condition in which a person has inflammation and swelling in the airways. Asthma patients also experience increased vasodilation in their lungs, i.e. the widening of blood vessels, which causes increased blood flow and results in increased inflammation. The goal of this project is to create a device that makes free standing hydrogel rings, modelling the structure of blood vessels, offering a simple approach to better understand asthma and potential treatments. The device used to form the rings is 3D printed and can be printed in a range of sizes. The rings are composed of collagen I that has been seeded with smooth muscle cells. Once the hydrogel rings are cast, they can be transferred to a 96 well plate and be free standing of any rigid structures. The ability to be free standing allows us to measure the ring diameter and wall thickness, as well as measure any change in size when a vasodilator is added. Future steps to be taken with this project include optimizing the size for biological relevance, introduce endothelial cells to create multiple layers of cells that are involved in signaling for vasodilation, and increase the responsiveness that the rings have to constriction factors as well as dilators.