High-Throughput Bioprinting of Geometrically-Controlled Pre-Vascularized Injectable Microgels for Accelerated Tissue Regeneration.

Successful integration of cell-laden tissue constructs with host vasculature depends on the presence of functional capillaries to provide oxygen and nutrients to the embedded cells. However, diffusion limitations of cell-laden biomaterials challenge regeneration of large tissue defects that require bulk-delivery of hydrogels and cells. Herein, a strategy to bioprint geometrically controlled, endothelial and stem-cell laden microgels in high-throughput is introduced, allowing these cells to form mature and functional pericyte-supported vascular capillaries in vitro, and then injecting these pre-vascularized constructs minimally invasively in-vivo. It is demonstrated that this approach offers both desired scalability for translational applications as well as unprecedented levels of control over multiple microgel parameters to design spatially-tailored microenvironments for better scaffold functionality and vasculature formation. As a proof-of-concept, the regenerative capacity of the bioprinted pre-vascularized microgels is compared with that of cell-laden monolithic hydrogels of the same cellular and matrix composition in hard-to-heal defects in vivo. The results demonstrate that the bioprinted microgels have faster and higher connective tissue formation, more vessels per area, and widespread presence of functional chimeric (human and murine) vascular capillaries across regenerated sites. The proposed strategy, therefore, addresses a significant issue in regenerative medicine, demonstrating a superior potential to facilitate translational regenerative efforts.

Impedance-Based Neutralizing Antibody Detection Biosensor with Application in SARS-CoV-2 Infection.

Although safe and efficacious coronavirus disease-2019 (COVID-19) vaccines are available, real protective immunity is revealed by the serum COVID-19 neutralizing antibody (NAb) concentration. NAbs deactivate the virus by attaching to the viral receptor-binding domain (RBD), which interacts with angiotensin-converting enzyme 2 (ACE2) on the human cell. This paper introduces inexpensive, rapid, sensitive, and quantifiable impedance-based immunosensors to evaluate the NAb. The sensor limit of detection is experimentally determined in different buffer dilutions using bovine IgG-anti-bovine IgG interaction. The dominance of AC electrokinetic transport and molecular diffusion in the sensor is investigated using scaling analysis and numerical simulations. The results demonstrated that the sensor detection mechanism is mainly based on the diffusion of the biomolecules onto the electrode surface. After evaluating the sensor working principles, viral RBD buffers, including different NAb concentrations, are applied to the sensor, immobilized with the human ACE2 (hACE2). Results demonstrate that the sensor is capable of NAb detection in the analytical measuring interval between 45 ng/mL and 185 ng/mL. Since the present sensor provides fast test results with lower costs, it can be used to assess the NAb in people's blood serum before receiving further COVID vaccine doses.

Vascular inflammation exposes perivascular cells to SARS-CoV-2 infection.

Pericytes stabilize blood vessels and promote vascular barrier function. However, vessels subjected to pro-inflammatory conditions have impaired barrier function, which has been suggested to potentially expose perivascular cells to SARS-CoV-2. To test this hypothesis, we engineered pericyte-supported vascular capillaries on-a-chip, and determined that the extravasation and binding of spike protein (S1) on perivascular cells of inflamed vessels to be significantly higher that in healthy controls, indicating a potential target to understand COVID-19 vascular complications.

A Microfluidic Dielectric Spectroscopy System for Characterization of Biological Cells in Physiological Media.

Dielectric spectroscopy (DS) is a promising cell screening method that can be used for diagnostic and drug discovery purposes. The primary challenge of using DS in physiological buffers is the electrode polarization (EP) that overwhelms the impedance signal within a large frequency range. These effects further amplify with the miniaturization of the measurement electrodes. In this study, we present a microfluidic system and the associated equivalent circuit models for real-time measurements of cell membrane capacitance and cytoplasm resistance in physiological buffers with 10 s increments. The current device captures several hundreds of biological cells in individual microwells through gravitational settling and measures the system's impedance using microelectrodes covered with dendritic gold nanostructures. Using PC-3 cells (a highly metastatic prostate cancer cell line) suspended in cell growth media (CGM), we demonstrate stable measurements of cell membrane capacitance and cytoplasm resistance in the device for over 15 min. We also describe a consistent application of the equivalent circuit model, starting from the reference measurements used to determine the system parameters. The circuit model is tested using devices with varying dimensions, and the obtained cell parameters between different devices are nearly identical. Further analyses of the impedance data have shown that accurate cell membrane capacitance and cytoplasm resistance can be extracted using a limited number of measurements in the 5 MHz to 10 MHz range. This will potentially reduce the timescale required for real-time DS measurements below 1 s. Overall, the new microfluidic device can be used for the dielectric characterization of biological cells in physiological buffers for various cell screening applications.

A dual-ink 3D printing strategy to engineer pre-vascularized bone scaffolds in-vitro.

A functional vascular supply is a key component of any large-scale tissue, providing support for the metabolic needs of tissue-remodeling cells. Although well-studied strategies exist to fabricate biomimetic scaffolds for bone regeneration, success rates for regeneration in larger defects can be improved by engineering microvascular capillaries within the scaffolds to enhance oxygen and nutrient supply to the core of the engineered tissue as it grows. Even though the role of calcium and phosphate has been well understood to enhance osteogenesis, it remains unclear whether calcium and phosphate may have a detrimental effect on the vasculogenic and angiogenic potential of endothelial cells cultured on 3D printed bone scaffolds. In this study, we presented a novel dual-ink bioprinting method to create vasculature interwoven inside CaP bone constructs. In this method, strands of a CaP ink and a sacrificial template material was used to form scaffolds containing CaP fibers and microchannels seeded with vascular endothelial and mesenchymal stem cells (MSCs) within a photo-crosslinkable gelatin methacryloyl (GelMA) hydrogel material. Our results show similar morphology of growing vessels in the presence of CaP bioink, and no significant difference in endothelial cell sprouting was found. Furthermore, our initial results showed the differentiation of hMSCs into pericytes in the presence of CaP ink. These results indicate the feasibility of creating vascularized bone scaffolds, which can be used for enhancing vascular formation in the core of bone scaffolds.

Bone-on-a-chip: microfluidic technologies and microphysiologic models of bone tissue.

Bone is an active organ that continuously undergoes an orchestrated process of remodeling throughout life. Bone tissue is uniquely capable of adapting to loading, hormonal, and other changes happening in the body, as well as repairing bone that becomes damaged to maintain tissue integrity. On the other hand, diseases such as osteoporosis and metastatic cancers disrupt normal bone homeostasis leading to compromised function. Historically, our ability to investigate processes related to either physiologic or diseased bone tissue has been limited by traditional models that fail to emulate the complexity of native bone. Organ-on-a-chip models are based on technological advances in tissue engineering and microfluidics, enabling the reproduction of key features specific to tissue microenvironments within a microfabricated device. Compared to conventional in-vitro and in-vivo bone models, microfluidic models, and especially organs-on-a-chip platforms, provide more biomimetic tissue culture conditions, with increased predictive power for clinical assays. In this review, we will report microfluidic and organ-on-a-chip technologies designed for understanding the biology of bone as well as bone-related diseases and treatments. Finally, we discuss the limitations of the current models and point toward future directions for microfluidics and organ-on-a-chip technologies in bone research.

Embedding cells within nanoscale, rapidly mineralizing hydrogels: A new paradigm to engineer cell-laden bone-like tissue.

Bone mineralization is a highly specific and dynamic nanoscale process that has been studied extensively from a structural, chemical, and biological standpoint. Bone tissue, therefore, may be defined by the interplay of its intricately mineralized matrix and the cells that regulate its biological function. However, the far majority of engineered bone model systems and bone replacement materials have been unable to replicate this key characteristic of bone tissue; that is, the ability of cells to be gradually and rapidly embedded in a three-dimensional (3D) heavily calcified matrix material. Here we review the characteristics that define the bone matrix from a nanostructural perspective. We then revisit the benefits and challenges of existing model systems and engineered bone replacement materials, and discuss recent efforts to replicate the biological, cellular, mechanical, and materials characteristics of bone tissue on the nano- to microscale. We pay particular attention to a recently proposed method developed by our group, which seeks to replicate key aspects of the entrapment of bone cells within a mineralized matrix with precisions down to the level of individual nano-crystallites, inclusive of the bone vasculature, and osteogenic differentiation process. In summary, this paper discusses existing and emerging evidence pointing towards future developments bridging the gap between the fields of biomineralization, structural biology, stem cells, and tissue engineering, which we believe will hold the key to engineer truly functional bone-like tissue in the laboratory.

3D Printing of Microgel-Loaded Modular Microcages as Instructive Scaffolds for Tissue Engineering.

Biomaterial scaffolds have served as the foundation of tissue engineering and regenerative medicine. However, scaffold systems are often difficult to scale in size or shape in order to fit defect-specific dimensions, and thus provide only limited spatiotemporal control of therapeutic delivery and host tissue responses. Here, a lithography-based 3D printing strategy is used to fabricate a novel miniaturized modular microcage scaffold system, which can be assembled and scaled manually with ease. Scalability is based on an intuitive concept of stacking modules, like conventional toy interlocking plastic blocks, allowing for literally thousands of potential geometric configurations, and without the need for specialized equipment. Moreover, the modular hollow-microcage design allows each unit to be loaded with biologic cargo of different compositions, thus enabling controllable and easy patterning of therapeutics within the material in 3D. In summary, the concept of miniaturized microcage designs with such straight-forward assembly and scalability, as well as controllable loading properties, is a flexible platform that can be extended to a wide range of materials for improved biological performance.

Bioinspired reconfiguration of 3D printed microfluidic hydrogels via automated manipulation of magnetic inks.

One of the key components in controlling fluid streams in microfluidic devices is the valve and gating modules. In most situations, these components are fixed at specific locations, and a new reconfiguration of microchannels requires costly and laborious fabrication of new devices. In this study, inspired by the human vasculature microcapillary reconfiguration in response to blood transport requirements, the idea of reconfigurable gel microfluidic systems is presented for the first time. A simple approach is described to print microchannels in methacrylated gelatin (GelMA) hydrogels by using agarose fibers that are loaded with iron microparticles. The agarose fibers can then be used as valves, which are then manipulated using a permanent magnet, providing the reconfigurability of the system. The feasibility of agarose gels is tested with different iron microparticle loadings as well as their resistance to fluid flows. Further, it is shown that using this technique, multiple configurations, as well as reconfigurability, are possible from a single device. This work opens the framework to design more intricate and reconfigurable microfluidic devices, which will decrease the cost and size of the final product.

Quantification of Cell Death Using an Impedance-Based Microfluidic Device.

Dielectric spectroscopy is a nondestructive method to characterize dielectric properties by measuring impedance data over a frequency spectrum. This method has been widely used for various applications such as counting, sizing, and monitoring biological cells and particles. Recently, utilization of this method has been suggested in various stages of the drug discovery process due to low sample consumption and fast analysis time. In this study, we used a previously developed microfluidic system to confine single PC-3 cells in microwells using dielectrophoretic forces and perform the impedance measurements. PC-3 cells are treated with 100 µM Enzalutamide drug, and their impedance response is recorded until the cells are totally dead as predicted with viability tests. Four different approaches are used to analyze the impedance spectrum. Equivalent circuit modeling is used to extract the cell electrical properties as a function of time. Principal component analysis (PCA) is used to quantify cellular response to drug as a function of time. Single frequency measurements are conducted to observe how the cells respond over time. Finally, opacity ratio is defined as an additional quantification method. This device is capable of quantitatively measuring drug effects on biological cells and detecting cell death. The results show that the proposed microfluidic system has the potential to be used in early stages of the drug discovery process.

Self-Similar Interfacial Impedance of Electrodes in High Conductivity Media: II. Disk Electrodes.

Electrode polarization effects were investigated using impedance spectroscopy measurements for planar and nanorod-structured gold disk electrodes at 100 Hz to 1 MHz frequency range and in 0.25 S/m to 1.5 S/m conductivity KCl solutions. Diameters of planar electrodes were varied from 50 µm to 2 mm to examine the effect of electrode size on impedance spectra. Normalizing the impedance magnitude with the spreading resistance and frequency with the characteristic time scale, all experimental data collapsed onto a universal curve, proving self-similarity. Experimental impedance results were compared well with that obtained from the numerical solution of Poisson-Nernst-Planck equations in axisymmetric domain. The influence of surface morphology was also investigated by generating cylindrical nanorods on a planar electrode. The 500 µm diameter electrode surface was covered with cylindrical nanorods with known height, diameter, and separation distance, which were characterized using scanning electron microscopy. The characteristic time scale for the nanorod-structured electrode increased by the surface enlargement factor obtained by cyclic voltammetry measurements. Self-similar interfacial impedance of electrodes was modeled using a constant phase element model. Current findings describe the coupled effects of electrode diameter, electrolyte conductivity, and electrode surface morphology on the impedance spectra of electrode/electrolyte system when the electric double layer between the nanorods does not overlap.

Electrical Impedance Measurements of Biological Cells in Response to External Stimuli.

Dielectric spectroscopy (DS) is a noninvasive technique for real-time measurements of the impedance spectra of biological cells. DS enables characterization of cellular dielectric properties such as membrane capacitance and cytoplasmic conductivity. We have developed a lab-on-a-chip device that uses an electro-activated microwells array for capturing, DS measurements, and unloading of biological cells. Impedance measurements were conducted at 0.2 V in the 10 kHz to 40 MHz range with 6 s time resolution. An equivalent circuit model was developed to extract the cell membrane capacitance and cell cytoplasmic conductivity from the impedance spectra. A human prostate cancer cell line, PC-3, was used to evaluate the device performance. Suspension of PC-3 cells in low conductivity buffers (LCB) enhanced their dielectrophoretic trapping and impedance response. We report the time course of the variations in dielectric properties of PC-3 cells suspended in LCB and their response to sudden pH change from a pH of 7.3 to a pH of 5.8. Importantly, we demonstrated that our device enabled real-time measurements of dielectric properties of live cancer cells and allowed the assessment of the cellular response to variations in buffer conductivity and pH. These data support further development of this device toward single cell measurements.

Self-Similar Interfacial Impedance of Electrodes in High Conductivity Media.

Electrode polarization (EP) happening due to accumulation of ions at the electrode/electrolyte interface is an inevitable phenomenon while measuring impedance spectrum in high conductivity buffers and at low RF spectrum. Well-characterized time scales elucidating the EP effect are important for the rational design of microfluidic devices and impedance sensors. In this Article, interfacial impedance at the electrode/electrolyte interface is investigated considering channel height and Debye length effects on characteristic time scale in a binary electrolyte solution using parallel plate electrode configuration. Experimental results reveal self-similarity of normalized electrical impedance as a function of the normalized frequency. The experimental results also match with numerical solutions obtained by finite element simulation of unsteady fully coupled Poisson-Nernst-Planck (PNP) equations. Furthermore, fractal shaped gold nanostructured electrodes are examined, and it has been proven that electric double layer (EDL) formed on porous electrode surfaces acts as a thick EDL and modifications to the characteristic time scale is necessary for porous electrodes. Finally, a constant phase element (CPE) model is proposed to account for the self-similar impedance spectrum, which can be used for different channel heights and solution conductivities.

Dielectrophoresis assisted loading and unloading of microwells for impedance spectroscopy.

Dielectric spectroscopy (DS) is a noninvasive, label-free, fast, and promising technique for measuring dielectric properties of biological cells in real time. We demonstrate a microchip that consists of electro-activated microwell arrays for positive dielectrophoresis assisted cell capture, DS measurements, and negative dielectrophoresis driven cell unloading; thus, providing a high-throughput cell analysis platform. To the best of our knowledge, this is the first microfluidic chip that combines electro-activated microwells and DS to analyze biological cells. Device performance is tested using Saccharomyces cerevisiae (yeast) cells. DEP response of yeast cells is determined by measuring their Clausius-Mossotti factor using biophysical models in parallel plate microelectrode geometry. This information is used to determine the excitation frequency to load and unload wells. Effect of yeast cells on the measured impedance spectrum was examined both experimentally and numerically. Good match between the numerical and experimental results establishes the potential use of the microchip device for extracting subcellular properties of biological cells in a rapid and nonexpensive manner.

Platinum black electrodeposited thread based electrodes for dielectrophoretic assembly of microparticles.

We report dielectrophoretic (DEP) assembly of biological cells and microparticles using platinum-black electrodeposited conductive textile fiber. The three-dimensional conductive structures with high aspect ratios were found to facilitate high electric field regions, as revealed by scanning electron microscope characterization. The effective conducting area (Aeff) and its stability of thread electrodes were estimated using electrochemical methods. Potential of platinum black electrodeposited thread as 3-D electrodes for creating high gradient electrical field for dielectrophoretic assembly of microspheres and Saccharomyces cerevisiae (yeast cells) into 1D and two-dimensional structures over long ranges under the application of low voltages (4-10 Vpp) has been demonstrated. The formation of highly ordered pearl chains of microparticles using thread electrodes when subjected to dielectrophoresis (DEP) has been discussed in detail.