Embedded Printing of Hydrogels and Watery Suspensions of Cells in Patterned Granular Baths.

Bioprinting within support media has emerged as the superior alternative to conventional extrusion printing. Not only because it allows for more freedom over the shapes that can be printed but also because it allows for the printing of inks that would not retain shape fidelity in freeform deposition such as watery liquids. Apart from functioning as mechanical support during embedded printing, hydrogel microparticle support media can provide the unique advantage of offering distinct chemotactic cues to cells printed in the baths by varying the composition of the hydrogel microparticles. There is great potential in compartmentalized granular baths consisting of different hydrogel particle materials in the field of tissue engineering, as these allow for the local inclusion of properties or cues to guide tissue development. In this work, we present a method to create compartmentalized embedding baths by printing multiple granular hydrogel materials that are widely used in tissue engineering. After adapting the volume fraction (φp) of the particles in the bath, we print within them using both inks composed of hydrogel or of cells and other particles suspended in watery liquid. Our process consists of the following three steps: First, the hydrogel microparticles are packed at a φp that allows them to be extruded while being reversibly jammed, facilitating the localized deposition of the granular media to form a compartmentalized bath. Second, each granular media is deposited in succession to create a packed suspension compartment, and by adding liquid post deposition, φp is reduced to allow for embedded printing. Finally, we demonstrate the printing of multiple inks within the compartmentalized embedding bath and highlight the distinct differences between using inks composed of hydrogels or inks composed of particles suspended in watery liquid. This approach combines the advantages of embedded printing through the use of granular media with the added ability to pattern multiple bioactive granular materials to locally affect the behavior of cells printed within the bath. We expect that this workflow will allow researchers to create spatially compartmentalized, customized bioactive embedding baths that allow for the embedded printing of inks composed of hydrogels, cells, and other particles adapted to their need.

Embedded 3D printing of dilute particle suspensions into dense complex tissue fibers using shear thinning xanthan baths.

In order to fabricate functional organoids and microtissues, a high cell density is generally required. As such, the placement of cell suspensions in molds or microwells to allow for cell concentration by sedimentation is the current standard for the production of organoids and microtissues. Even though molds offer some level of control over the shape of the resulting microtissue, this control is limited as microtissues tend to compact towards a sphere after sedimentation of the cells. 3D bioprinting on the other hand offers complete control over the shape of the resulting structure. Even though the printing of dense cell suspensions in the ink has been reported, extruding dense cellular suspensions is challenging and generally results in high shear stresses on the cells and a poor shape fidelity of the print. As such, additional materials such as hydrogels are added in the bioink to limit shear stresses, and to improve shape fidelity and resolution. The maximum cell concentration that can be incorporated in a hydrogel-based ink before the ink's rheological properties are compromised, is significantly lower than the concentration in a tissue equivalent. Additionally, the hydrogel components often interfere with cellular self-assembly processes. To circumvent these limitations, we report a simple and inexpensive xanthan bath based embedded printing method to 3D print dense functional linear tissues using dilute particle suspensions consisting of cells, spheroids, hydrogel beads, or combinations thereof. Using this method, we demonstrated the self-organization of functional cardiac tissue fibers with a layer of epicardial cells surrounding a body of cardiomyocytes.

Assessment of flow within developing chicken vasculature and biofabricated vascularized tissues using multimodal imaging techniques.

Fluid flow shear stresses are strong regulators for directing the organization of vascular networks. Knowledge of structural and flow dynamics information within complex vasculature is essential for tuning the vascular organization within engineered tissues, by manipulating flows. However, reported investigations of vascular organization and their associated flow dynamics within complex vasculature over time are limited, due to limitations in the available physiological pre-clinical models, and the optical inaccessibility and aseptic nature of these models. Here, we developed laser speckle contrast imaging (LSCI) and side-stream dark field microscopy (SDF) systems to map the vascular organization, spatio-temporal blood flow fluctuations as well as erythrocytes movements within individual blood vessels of developing chick embryo, cultured within an artificial eggshell system. By combining imaging data and computational simulations, we estimated fluid flow shear stresses within multiscale vasculature of varying complexity. Furthermore, we demonstrated the LSCI compatibility with bioengineered perfusable muscle tissue constructs, fabricated via molding techniques. The presented application of LSCI and SDF on perfusable tissues enables us to study the flow perfusion effects in a non-invasive fashion. The gained knowledge can help to use fluid perfusion in order to tune and control multiscale vascular organization within engineered tissues.

Ultrahigh-Throughput Production of Monodisperse and Multifunctional Janus Microparticles Using in-Air Microfluidics.

Compartmentalized Janus microparticles advance many applications ranging from chemical synthesis to consumer electronics. Although these particles can be accurately manufactured using microfluidic droplet generators, the per-nozzle throughputs are relatively low (∼µL/min). Here, we use "in-air microfluidics" to combine liquid microjets in midair, thereby enabling orders of magnitude faster production of Janus microparticles (∼mL/min) as compared to chip-based microfluidics. Monodisperse Janus microparticles with diameters between 50 and 500 µm, tunable compartment sizes, and functional cargo are controllably produced. Furthermore, these microparticles are designed as magnetically steerable microreactors, which represents a novel tool to perform enzymatic cascade reactions within continuous fluid flows.

Multicompartment Artificial Organelles Conducting Enzymatic Cascade Reactions inside Cells.

Cell organelles are subcellular structures entrapping a set of enzymes to achieve a specific functionality. The incorporation of artificial organelles into cells is a novel medical paradigm which might contribute to the treatment of various cell disorders by replacing malfunctioning organelles. In particular, artificial organelles are expected to be a powerful solution in the context of enzyme replacement therapy since enzymatic malfunction is the primary cause of organelle dysfunction. Although several attempts have been made to encapsulate enzymes within a carrier vehicle, only few intracellularly active artificial organelles have been reported to date and they all consist of single-compartment carriers. However, it is noted that biological organelles consist of multicompartment architectures where enzymatic reactions are executed within distinct subcompartments. Compartmentalization allows for multiple processes to take place in close vicinity and in a parallel manner without the risk of interference or degradation. Here, we report on a subcompartmentalized and intracellularly active carrier, a crucial step for advancing artificial organelles. In particular, we develop and characterize a novel capsosome system, which consists of multiple liposomes and fluorescent gold nanoclusters embedded within a polymer carrier capsule. We subsequently demonstrate that encapsulated enzymes preserve their activity intracellularly, allowing for controlled enzymatic cascade reaction within a host cell.