CELLNET technology: Spatially organized, functional 3D networks at single cell resolution.

Cells possess the remarkable ability to generate tissue-specific 3D interconnected networks and respond to a wide range of stimuli. Understanding the link between the spatial arrangement of individual cells and their networks' emergent properties is necessary for the discovery of both fundamental biology as well as applied therapeutics. However, current methods spanning from lithography to 3D photo-patterning to acoustofluidic devices are unable to generate interconnected and organized single cell 3D networks within native extracellular matrix (ECM). To address this challenge, we report a novel technology coined as CELLNET. This involves the generation of crosslinked collagen within multi-chambered microfluidic devices followed by femtosecond laser ablation of 3D microchannel networks and cell seeding. Using model cells, we show that cell migrate within ablated networks within hours, self-organize and form viable, interconnected, 3D networks in custom architectures such as square grid, concentric circle, parallel lines, and spiral patterns. Heterotypic CELLNETs can also be generated by seeding multiple cell types in side-chambers of the devices. The functionality of cell networks can be studied by monitoring the real-time calcium signaling response of individual cells and signal propagation within CELLNETs when subjected to flow stimulus alone or a sequential combination of flow and biochemical stimuli. Furthermore, user-defined disrupted CELLNETs can be generated by lethally injuring target cells within the 3D network and analyzing the changes in their signaling dynamics. As compared to the current self-assembly based methods that exhibit high variability and poor reproducibility, CELLNETs can generate organized 3D single-cell networks and their real-time signaling responses to a range of stimuli can be accurately captured using simple cell seeding and easy-to-handle microfluidic devices. CELLNET, a new technology agnostic of cell types, ECM formulations, 3D cell-connectivity designs, or location and timing of network disruptions, could pave the way to address a range of fundamental and applied bioscience applications. Teaser: New technology to generate 3D single cell interconnected and disrupted networks within natural extracellular matrix in custom configurations.

Droplet bioprinting of acellular and cell-laden structures at high-resolutions.

Advances in digital light projection(DLP) based (bio) printers have made printing of intricate structures at high resolution possible using a wide range of photosensitive bioinks. A typical setup of a DLP bioprinter includes a vat or reservoir filled with liquid bioink, which presents challenges in terms of cost associated with bioink synthesis, high waste, and gravity-induced cell settling, contaminations, or variation in bioink viscosity during the printing process. Here, we report a vat-free, low-volume, waste-free droplet bioprinting method capable of rapidly printing 3D soft structures at high resolution using model bioinks and model cells. A multiphase many-body dissipative particle dynamics model was developed to simulate the dynamic process of droplet-based DLP printing and elucidate the roles of surface wettability and bioink viscosity. Process variables such as light intensity, photo-initiator concentration, and bioink formulations were optimized to print 3D soft structures (∼0.4-3 kPa) with a typical layer thickness of 50µm, an XY resolution of 38 ± 1.5µm and Z resolution of 237 ± 5.4µm. To demonstrate its versatility, droplet bioprinting was used to print a range of acellular 3D structures such as a lattice cube, a Mayan pyramid, a heart-shaped structure, and a microfluidic chip with endothelialized channels. Droplet bioprinting, performed using model C3H/10T1/2 cells, exhibited high viability (90%) and cell spreading. Additionally, microfluidic devices with internal channel networks lined with endothelial cells showed robust monolayer formation while osteoblast-laden constructs showed mineral deposition upon osteogenic induction. Overall, droplet bioprinting could be a low-cost, no-waste, easy-to-use, method to make customized bioprinted constructs for a range of biomedical applications.

Vimentin promotes collective cell migration through collagen networks via increased matrix remodeling and spheroid fluidity.

The intermediate filament (IF) protein vimentin is associated with many diseases with phenotypes of enhanced cellular migration and aggressive invasion through the extracellular matrix (ECM) of tissues, but vimentin's role in in-vivo cell migration is still largely unclear. Vimentin is important for proper cellular adhesion and force generation, which are critical to cell migration; yet the vimentin cytoskeleton also hinders the ability of cells to squeeze through small pores in ECM, resisting migration. To identify the role of vimentin in collective cell migration, we generate spheroids of wide-type and vimentin-null mouse embryonic fibroblasts (mEFs) and embed them in a 3D collagen matrix. We find that loss of vimentin significantly impairs the ability of the spheroid to collectively expand through collagen networks and remodel the collagen network. Traction force analysis reveals that vimentin null spheroids exert less contractile force than their wild-type counterparts. In addition, spheroids made of mEFs with only vimentin unit length filaments (ULFs) exhibit similar behavior as vimentin-null spheroids, suggesting filamentous vimentin is required to promote 3D collective cell migration. We find the vimentin-mediated collective cell expansion is dependent on matrix metalloproteinase (MMP) degradation of the collagen matrix. Further, 3D vertex model simulation of spheroid and embedded ECM indicates that wild-type spheroids behave more fluid-like, enabling more active pulling and reconstructing the surrounding collagen network. Altogether, these results signify that VIF plays a critical role in enhancing migratory persistence in 3D matrix environments through MMP transportation and tissue fluidity.

Droplet bioprinting of acellular and cell-laden structures at high-resolutions.

Advances in Digital Light Processing (DLP) based (bio) printers have made printing of intricate structures at high resolution possible using a wide range of photosensitive bioinks. A typical setup of a DLP bioprinter includes a vat or reservoir filled with liquid bioink, which presents challenges in terms of cost associated with bioink synthesis, high waste, and gravity-induced cell settling, contaminations, or variation in bioink viscosity during the printing process. Here, we report a vat-free, low-volume, waste-free droplet bioprinting method capable of rapidly printing 3D soft structures at high resolution using model bioinks. A multiphase many-body dissipative particle dynamics (mDPD) model was developed to simulate the dynamic process of droplet-based DLP printing and elucidate the roles of surface wettability and bioink viscosity. Process variables such as light intensity, photo-initiator concentration, and bioink formulations were optimized to print 3D soft structures (∼0.4 to 3 kPa) with an XY resolution of 38 ± 1.5 µm and Z resolution of 237±5.4 µm. To demonstrate its versatility, droplet bioprinting was used to print a range of acellular 3D structures such as a lattice cube, a Mayan pyramid, a heart-shaped structure, and a microfluidic chip with endothelialized channels. Droplet bioprinting, performed using model C3H/10T1/2 cells, exhibited high viability (90%) and cell spreading. Additionally, microfluidic devices with internal channel network lined with endothelial cells showed robust monolayer formation while osteoblast-laden constructs showed mineral deposition upon osteogenic induction. Overall, droplet bioprinting could be a low-cost, no-waste, easy-to-use, method to make customized bioprinted constructs for a range of biomedical applications.

Printing Double-Network Tough Hydrogels Using Temperature-Controlled Projection Stereolithography (TOPS).

We report a new method to shape double-network (DN) hydrogels into customized 3D structures that exhibit superior mechanical properties in both tension and compression. A one-pot prepolymer formulation containing photo-cross-linkable acrylamide and thermoreversible sol-gel κ-carrageenan with a suitable cross-linker and photoinitiators/absorbers is optimized. A new TOPS system is utilized to photopolymerize the primary acrylamide network into a 3D structure above the sol-gel transition of κ-carrageenan (80 °C), while cooling down generates the secondary physical κ-carrageenan network to realize tough DN hydrogel structures. 3D structures, printed with high lateral (37 µm) and vertical (180 µm) resolutions and superior 3D design freedoms (internal voids), exhibit ultimate stress and strain of 200 kPa and 2400%, respectively, under tension and simultaneously exhibit a high compression stress of 15 MPa with a strain of 95%, both with high recovery rates. The roles of swelling, necking, self-healing, cyclic loading, dehydration, and rehydration on the mechanical properties of printed structures are also investigated. To demonstrate the potential of this technology to make mechanically reconfigurable flexible devices, we print an axicon lens and show that a Bessel beam can be dynamically tuned via user-defined tensile stretching of the device. This technique can be broadly applied to other hydrogels to make novel smart multifunctional devices for a range of applications.

Feature-based volumetric defect classification in metal additive manufacturing.

Volumetric defect types commonly observed in the additively manufactured parts differ in their morphologies ascribed to their formation mechanisms. Using high-resolution X-ray computed tomography, this study analyzes the morphological features of volumetric defects, and their statistical distribution, in laser powder bed fused Ti-6Al-4V. The geometries of three common types of volumetric defects; i.e., lack of fusions, gas-entrapped pores, and keyholes, are quantified by nine parameters including maximum dimension, roundness, sparseness, aspect ratio, and more. It is shown that the three defect types share overlaps of different degrees in the ranges of their morphological parameters; thus, employing only one or two parameters cannot uniquely determine a defect's type. To overcome this challenge, a defect classification methodology incorporating multiple morphological parameters has been proposed. In this work, by employing the most discriminating parameters, this methodology has been shown effective when implemented into decision tree (>98% accuracy) and artificial neural network (>99% accuracy).

Femtosecond Laser Densification of Hydrogels to Generate Customized Volume Diffractive Gratings.

Inspired by nature's ability to shape soft biological materials to exhibit a range of optical functionalities, we report femtosecond (fs) laser-induced densification as a new method to generate volume or subsurface diffractive gratings within ordinary hydrogel materials. We characterize the processing range in terms of fs laser power, speed, and penetration depths for achieving densification within poly(ethylene glycol) diacrylate (PEGDA) hydrogel and characterize the associated change in local refractive index (RI). The RI change facilitates the fabrication of custom volume gratings (parallel line, grid, square, and ring gratings) within PEGDA. To demonstrate this method's broad applicability, fs laser densification was used to generate line gratings within the phenylboronic acid (PBA) hydrogel, which is known to be responsive to changes in pH. In the future, this technique can be used to convert ordinary hydrogels into multicomponent biophotonic systems.