Role of temperature on bio-printability of gelatin methacryloyl bioink in two-step cross-linking strategy for tissue engineering applications.

Additive manufacturing has shown promising results in reconstructing three-dimensional (3D) living tissues for various applications, including tissue engineering, regenerative medicine, drug discovery, and high-throughput drug screening. In extrusion-based bioprinters, stable formation of filaments and high-fidelity deposition of bioinks are the primary challenges in fabrication of physiologically relevant tissue constructs. Among various bioinks, gelatin methacryloyl (GelMA) is known as a photocurable and physicochemically tunable hydrogel with a demonstrated biocompatibility and tunable biodegradation properties. The two-step crosslinking of GelMA (reversible thermal gelation and permanent photo-crosslinking) has attracted researchers to make complex tissue constructs. Despite promising results in filament formation and printability of this hydrogel, the effect of temperature on physicochemical properties, cytocompatibility, and biodegradation of the hydrogel are to be investigated. This work studies the effect of thermoreversible, physical crosslinking on printability of GelMA. The results of 3D printing of GelMA at different temperatures followed by irreversible chemical photo-crosslinking show that the decrease in temperature improves the filament formation and shape fidelity of the deposited hydrogel, particularly at the temperatures around 15 °C. Time dependant mechanical testing of the printed samples revealed that decreasing the extruding temperature increases the elastic properties of the extruded filaments. Furthermore, our novel approach in minimizing the slippage effect during rheological study enabled to measure changes in linear and non-linear viscoelastic properties of the printed samples at different temperatures. A considerable increase in storage modulus of the extruded samples printed at lower temperatures confirms their higher solid behavior. Scanning electron microscopy revealed a remarkable decrease in porosity of the extruded hydrogels by decreasing the temperature. Chemical analysis by Fourier-transform infrared spectroscopy and circular dichroism showed a direct relationship between the coil-helix transition in hydrogel macromers and its physical alterations. Finally, biodegradation and cytocompatibility of the extruded hydrogels decreased at lower extruding temperatures.

Picoliter agar droplet breakup in microfluidics meets microbiology application: numerical and experimental approaches.

Droplet microfluidics has provided lab-on-a-chip platforms with the capability of bacteria encapsulation in biomaterials, controlled culture environments, and live monitoring of growth and proliferation. The droplets are mainly generated from biomaterials with temperature dependent gelation behavior which necessitates stable and size-controlled droplet formation within microfluidics. Here, the biomaterial is agar hydrogel with a non-Newtonian response at operating temperatures below 40 °C, the upper-temperature threshold for cells and pathogens. The size of the produced droplets and the formation regimes are examined when the agar is injected at a constant temperature of 37 °C with agar concentrations of 0.5%, 1%, and 2% and different flow rate ratios of the dispersed phase to the continuous phase (φ: 0.1 to 1). The numerical simulations show that φ and the capillary number (Ca) are the key parameters controlling the agar droplet size and formation regime, from dripping to jetting. Also, increasing the agar concentration produces smaller droplets. The simulation data were validated against experimental agar droplet generation and transport in microfluidics. This work helps to understand the physics of droplet generation in droplet microfluidic systems operating with non-Newtonian fluids. Pathogenic bacteria were successfully cultured and monitored in high resolution in agar droplets for further research in antibiotic susceptibility testing in bacteremia and urinary tract infection.

Engineering Shelf-Stable Coating for Microfluidic Organ-on-a-Chip Using Bioinspired Catecholamine Polymers.

The conceptualization of body-on-a-chip in 2004 resulted in a new approach for studying human physiology in three-dimensional culture. Despite pioneering works and the progress made in replicating human physiology on-a-chip, the stability, reliability, and preservation of cell-culture-treated microfluidic chips remain a challenge. The development of a reliable surface treatment technique to more efficiently and reproducibly modify microfluidic channels would significantly simplify the process of creating and implementing organ-on-a-chip (OOC) systems. In this work, a new flow-based coating technique using bioinspired polymers was implemented to create reliable, reproducible, ready-to-use microfluidic cell culture chips for OOC studies. Single-channel polydimethylsiloxane microfluidic chips were coated with the bioinspired catecholamine polymers, polydopamine (PDA) and polynorepinephrine (PNE), using a flow-based coating technique. The functionality of the resulting microfluidic chips was evaluated by extensive surface characterizations, at 130 °C, in the presence of various cleaning and culture media in static and flow conditions regularly used in OOCs and tested for shelf life by storing the coated microfluidic chips for 4 months at room temperature. Microfluidic chips coated with polycatecholamine were then seeded with the mouse cancer cell line Cath.a.differentiated (CAD) and with the normal human cerebral microvascular endothelial cell line human cerebral microvascular endothelial cells (hCMEC)/D3. Cell viability, cell phenotype, and cell functionality were assessed to evaluate the performance of both the coatings and the surface treatment technique. Both PDA- and PNE-coated microfluidic chips maintained high viability, phenotype, and functionality of CAD cells and hCMEC/D3 cells. In addition, CAD cells retained high viability when they were cultured in both the polymer-coated chips, which were stored at room temperature for up to 120 days. These results suggest that flow-based techniques to coat surfaces with polycatecholamines can be used to generate ready-to-use microfluidic OOC chips that offer long-term stability and reliability for the culture of cell types with application in pathophysiological studies and drug screening.

Rapid and Highly Controlled Generation of Monodisperse Multiple Emulsions via a One-Step Hybrid Microfluidic Device.

Multiple Emulsions (MEs) contain a drop laden with many micro-droplets. A single-step microfluidic-based synthesis process of MEs is presented to provide a rapid and controlled generation of monodisperse MEs. The design relies on the interaction of three immiscible fluids with each other in subsequent droplet formation steps to generate monodisperse ME constructs. The design is within a microchannel consists of two compartments of cross-junction and T-junction. The high shear stress at the cross-junction creates a stagnation point that splits the first immiscible phase to four jet streams each of which are sprayed to micrometer droplets surrounded by the second phase. The resulted structure is then supported by the third phase at the T-junction to generate and transport MEs. The ME formation within microfluidics is numerically simulated and the effects of several key parameters on properties of MEs are investigated. The dimensionless modeling of ME formation enables to change only one parameter at the time and analyze the sensitivity of the system to each parameter. The results demonstrate the capability of highly controlled and high-throughput MEs formation in a one-step synthesis process. The consecutive MEs are monodisperse in size which open avenues for the generation of controlled MEs for different applications.

Passive microinjection within high-throughput microfluidics for controlled actuation of droplets and cells.

Microinjection is an effective actuation technique used for precise delivery of molecules and cells into droplets or controlled delivery of genes, molecules, proteins, and viruses into single cells. Several microinjection techniques have been developed for actuating droplets and cells. However, they are still time-consuming, have shown limited success, and are not compatible with the needs of high-throughput (HT) serial microinjection. We present a new passive microinjection technique relying on pressure-driven fluid flow and pulsative flow patterns within an HT droplet microfluidic system to produce serial droplets and manage rapid and highly controlled microinjection into droplets. A microneedle is secured within the injection station to confine droplets during the microinjection. The confinement of droplets on the injection station prevents their movement or deformation during the injection process. Three-dimensional (3D) computational analysis is developed and validated to model the dynamics of multiphase flows during the emulsion generation. We investigate the influence of pulsative flows, microneedle parameters and synchronization on the efficacy of microinjection. Finally, the feasibility of implementing our microinjection model is examined experimentally. This technique can be used for tissue engineering, cells actuation and drug discovery as well as developing new strategies for drug delivery.