An embedded microfluidic valve for dynamic control of cellular communication.

The communication between different cell populations is an important aspect of many natural phenomena that can be studied with microfluidics. Using microfluidic valves, these complex interactions can be studied with a higher level of control by placing a valve between physically separated populations. However, most current valve designs do not display the properties necessary for this type of system, such as providing variable flow rate when embedded inside a microfluidic device. While some valves have been shown to have such tunable behavior, they have not been used for dynamic, real-time outputs. We present an electric solenoid valve that can be fabricated completely outside of a cleanroom and placed into any microfluidic device to offer control of dynamic fluid flow rates and profiles. After characterizing the behavior of this valve under controlled test conditions, we developed a regression model to determine the required input electrical signal to provide the solenoid the ability to create a desired flow profile. With this model, we demonstrated that the valve could be controlled to replicate a desired, time-varying pattern for the interface position of a co-laminar fluid stream. Our approach can be performed by other investigators with their microfluidic devices to produce predictable, dynamic fluidic behavior. In addition to modulating fluid flows, this work will be impactful for controlling cellular communication between distinct populations or even chemical reactions occurring in microfluidic channels.

Nanotopographical Cues Tune the Therapeutic Potential of Extracellular Vesicles for the Treatment of Aged Skeletal Muscle Injuries.

Skeletal muscle regeneration relies on the tightly temporally regulated lineage progression of muscle stem/progenitor cells (MPCs) from activation to proliferation and, finally, differentiation. However, with aging, MPC lineage progression is disrupted and delayed, ultimately causing impaired muscle regeneration. Extracellular vesicles (EVs) have attracted broad attention as next-generation therapeutics for promoting tissue regeneration. As a next step toward clinical translation, strategies to manipulate EV effects on downstream cellular targets are needed. Here, we developed an engineering strategy to tune the therapeutic potential of EVs using nanotopographical cues. We found that EVs released by young MPCs cultured on flat substrates (fEVs) promoted the proliferation of aged MPCs while EVs released by MPCs cultured on nanogratings (nEVs) promoted myogenic differentiation. We then employed a bioengineered 3D muscle aging model to optimize the administration protocol and test the therapeutic potential of fEVs and nEVs in a high-throughput manner. We found that the sequential administration first of fEVs during the phase of MPC proliferative expansion (i.e., 1 day after injury) followed by nEV administration at the stage of MPC differentiation (i.e., 3 days after injury) enhanced aged muscle regeneration to a significantly greater extent than fEVs and nEVs delivered either in isolation or mixed. The beneficial effects of the sequential EV treatment strategy were further validated in vivo, as evidenced by increased myofiber size and improved functional recovery. Collectively, our study demonstrates the ability of topographical cues to tune EV therapeutic potential and highlights the importance of optimizing the EV administration strategy to accelerate aged skeletal muscle regeneration.

Characterization of neural mechanotransduction response in human traumatic brain injury organoid model.

The ability to model physiological systems through 3D neural in-vitro systems may enable new treatments for various diseases while lowering the need for challenging animal and human testing. Creating such an environment, and even more impactful, one that mimics human brain tissue under mechanical stimulation, would be extremely useful to study a range of human-specific biological processes and conditions related to brain trauma. One approach is to use human cerebral organoids (hCOs) in-vitro models. hCOs recreate key cytoarchitectural features of the human brain, distinguishing themselves from more traditional 2D cultures and organ-on-a-chip models, as well as in-vivo animal models. Here, we propose a novel approach to emulate mild and moderate traumatic brain injury (TBI) using hCOs that undergo strain rates indicative of TBI. We subjected the hCOs to mild (2 s[Formula: see text]) and moderate (14 s[Formula: see text]) loading conditions, examined the mechanotransduction response, and investigated downstream genomic effects and regulatory pathways. The revealed pathways of note were cell death and metabolic and biosynthetic pathways implicating genes such as CARD9, ENO1, and FOXP3, respectively. Additionally, we show a steeper ascent in calcium signaling as we imposed higher loading conditions on the organoids. The elucidation of neural response to mechanical stimulation in reliable human cerebral organoid models gives insights into a better understanding of TBI in humans.

Freeform 3D Ice Printing (3D-ICE) at the Micro Scale.

Water is one of the most important elements for life on earth. Water's rapid phase-change ability along with its environmental and biological compatibility also makes it a unique structural material for 3D printing of ice structures reproducibly and accurately. This work introduces the freeform 3D ice printing (3D-ICE) process for high-speed and reproducible fabrication of ice structures with micro-scale resolution. Drop-on-demand deposition of water onto a -35 °C platform rapidly transforms water into ice. The dimension and geometry of the structures are critically controlled by droplet ejection frequency modulation and stage motions. The freeform approach obviates layer-by-layer construction and support structures, even for overhang geometries. Complex and overhang geometries, branched hierarchical structures with smooth transitions, circular cross-sections, smooth surfaces, and micro-scale features (as small as 50 µm) are demonstrated. As a sample application, the ice templates are used as sacrificial geometries to produce resin parts with well-defined internal features. This approach could bring exciting opportunities for microfluidics, biomedical devices, soft electronics, and art.

Inducing Vascular Grammars for Anomaly Classification in Brain Angiograms.

As machine learning is used to make strides in medical diagnostics, few methods provide heuristics from which human doctors can learn directly. This work introduces a method for leveraging human observable structures, such as macroscale vascular formations, for producing assessments of medical conditions with relatively few training cases, and uncovering patterns that are potential diagnostic aids. The approach draws on shape grammars, a rule-based technique, pioneered in design and architecture, and accelerated through a recursive subgraph mining algorithm. The distribution of rule instances in the data from which they are induced is then used as an intermediary representation enabling common classification and anomaly detection approaches to identify indicative rules with relatively small data sets. The method is applied to seven-tesla time-of-flight angiography MRI (n = 54) of human brain vasculature. The data were segmented and induced to generate representative grammar rules. Ensembles of rules were isolated to implicate vascular conditions reliably. This application demonstrates the power of automated structured intermediary representations for assessing nuanced biological form relationships, and the strength of shape grammars, in particular for identifying indicative patterns in complex vascular networks.

Cell alignment modulated by surface nano-topography - Roles of cell-matrix and cell-cell interactions.

The propensity of cells to align in particular directions is relevant to a number of areas, including tissue engineering and biohybrid robotics. Cell alignment is modulated through various extracellular conditions including surface topographies, mechanical cues from cell-matrix interactions, and cell-cell interactions. Understanding of these conditions provides guidance for desirable cellular structure constructions. In this study, we examine the roles of surface topographies and cell-cell interactions in inducing cell alignment. We employed wavy surface topographies at the nanometer scale as a model extracellular environment for cell culture. The results show that, within a certain range of wavelengths and amplitudes of the surface topographies, cell alignment is dependent on cell confluency. This dependence on both topology and confluency suggests interplay between cell-cell and cell-matrix interactions in inducing cell alignment. Images of sparsely distributed and confluent cells also demonstrated clear differences in the structures of their focal adhesion complexes. To understand this effect, we introduced anti-N-cadherin to cell culture to inhibit cell-cell interactions. The results show that, when anti-N-cadherin was applied, cells on wavy surfaces required greater confluency to achieve the same alignment compared to that in the absence of anti-N-cadherin. The understanding of the cell alignment mechanisms will be important in numerous potential applications such as scaffold design, tissue repair, and development of biohybrid robotic systems. STATEMENT OF SIGNIFICANCE: Cell alignment plays a critical role in numerous biological functions. Advances in tissue engineering utilizes cell alignment to restore, maintain, or even replace different types of biological tissues. The clinical impact that tissue engineering has made is facilitated by advancements in the understanding of interactions between scaffolds, biological factors, and cells. This work further elucidates the role of cell-cell interactions in promoting the organization of biological tissues.

Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System.

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such as aspiration or coverslip compression have been used to mechanically stimulate whole embryos. These approaches limit experimental design as they are imprecise, require manual handling, and can process only a couple of embryos simultaneously. Microfluidic systems have great potential for automating such experimental tasks while increasing throughput and precision. This article describes a microfluidic system developed to precisely compress whole Drosophila melanogaster (fruit fly) embryos. This system features microchannels with pneumatically actuated deformable sidewalls and enables embryo alignment, immobilization, compression, and post-stimulation collection. By parallelizing these microchannels into seven lanes, steady or dynamic compression patterns can be applied to hundreds of Drosophila embryos simultaneously. Fabricating this system on a glass coverslip facilitates the simultaneous mechanical stimulation and imaging of samples with high-resolution microscopes. Moreover, the utilization of biocompatible materials, like PDMS, and the ability to flow fluid through the system make this device capable of long-term experiments with media-dependent samples. This approach also eliminates the requirement for manual mounting which mechanically stresses samples. Furthermore, the ability to quickly collect samples from the microchannels enables post-stimulation analyses, including -omics assays which require large sample numbers unattainable using traditional mechanical stimulation approaches. The geometry of this system is readily scalable to different biological systems, enabling numerous fields to benefit from the functional features described herein including high sample throughput, mechanical stimulation or immobilization, and automated alignment.

3D Collagen Vascular Tumor-on-a-Chip Mimetics for Dynamic Combinatorial Drug Screening.

Disease models, including in vitro cell culture and animal models, have contributed significantly to developing diagnostics and treatments over the past several decades. The successes of traditional drug screening methods were generally hampered by not adequately mimicking critical in vivo features, such as a 3D microenvironment and dynamic drug diffusion through the extracellular matrix (ECM). To address these issues, we developed a 3D dynamic drug delivery system for cancer drug screening that mimicks drug dissemination through the tumor vasculature and the ECM by creating collagen-embedded microfluidic channels. Using this novel 3D ECM microsystem, we compared viability of tumor pieces with traditionally used 2D methods in response to three different drug combinations. Drug diffusion profiles were evaluated by simulation methods and tested in the 3D ECM microsystem and a 2D 96-well setup. Compared with the 2D control, the 3D ECM microsystem produced reliable data on viability, drug ratios, and combination indeces. This novel approach enables higher throughput and sets the stage for future applications utilizing drug sensitivity predicting algorithms based on dynamic diffusion profiles requiring only minimal patient tissue. Our findings moved drug sensitivity screening closer to clinical implications with a focus on testing combinatorial drug effects, an option often limited by the amount of available patient tissues.

Hierarchical Machine Learning for High-Fidelity 3D Printed Biopolymers.

A hierarchical machine learning (HML) framework is presented that uses a small dataset to learn and predict the dominant build parameters necessary to print high-fidelity 3D features of alginate hydrogels. We examine the 3D printing of soft hydrogel forms printed with the freeform reversible embedding of suspended hydrogel method based on a CAD file that isolated the single-strand diameter and shape fidelity of printed alginate. Combinations of system variables ranging from print speed, flow rate, ink concentration to nozzle diameter were systematically varied to generate a small dataset of 48 prints. Prints were imaged and scored according to their dimensional similarity to the CAD file, and high print fidelity was defined as prints with less than 10% error from the CAD file. As a part of the HML framework, statistical inference was performed, using the least absolute shrinkage and selection operator to find the dominant variables that drive the error in the final prints. Model fit between the system parameters and print score was elucidated and improved by a parameterized middle layer of variable relationships which showed good performance between the predicted and observed data (R2 = 0.643). Optimization allowed for the prediction of build parameters that gave rise to high-fidelity prints of the measured features. A trade-off was identified when optimizing for the fidelity of different features printed within the same construct, showing the need for complex predictive design tools. A combination of known and discovered relationships was used to generate process maps for the 3D bioprinting designer that show error minimums based on the chosen input variables. Our approach offers a promising pathway toward scaling 3D bioprinting by optimizing print fidelity via learned build parameters that reduce the need for iterative testing.

Toward Vasculature in Skeletal Muscle-on-a-Chip through Thermo-Responsive Sacrificial Templates.

Developing new approaches for vascularizing synthetic tissue systems will have a tremendous impact in diverse areas. One area where this is particularly important is developing new skeletal muscle tissue systems, which could be utilized in physiological model studies and tissue regeneration. To develop vascularized approaches a microfluidic on-chip design for creating channels in polymer systems can be pursued. Current microfluidic tissue engineering methods include soft lithography, rapid prototyping, and cell printing; however, these have limitations such as having their scaffolding being inorganic, less desirable planar vasculature geometry, low fabrication efficiency, and limited resolution. Here we successfully developed a circular microfluidic channel embedded in a 3D extracellular matrix scaffolding with 3D myogenesis. We used a thermo-responsive polymer approach with micromilling-molding and designed a mixture of polyester wax and paraffin wax to fabricate the sacrificial template for microfluidic channel generation in the scaffolding. These findings will impact a number of fields including biomaterials, biomimetic structures, and personalized medicine in the future.

Probing coordinated co-culture cancer related motility through differential micro-compartmentalized elastic substrates.

Cell development and behavior are driven by internal genetic programming, but the external microenvironment is increasingly recognized as a significant factor in cell differentiation, migration, and in the case of cancer, metastatic progression. Yet it remains unclear how the microenvironment influences cell processes, especially when examining cell motility. One factor that affects cell motility is cell mechanics, which is known to be related to substrate stiffness. Examining how cells interact with each other in response to mechanically differential substrates would allow an increased understanding of their coordinated cell motility. In order to probe the effect of substrate stiffness on tumor related cells in greater detail, we created hard-soft-hard (HSH) polydimethylsiloxane (PDMS) substrates with alternating regions of different stiffness (200 and 800 kPa). We then cultured WI-38 fibroblasts and A549 epithelial cells to probe their motile response to the substrates. We found that when the 2 cell types were exposed simultaneously to the same substrate, fibroblasts moved at an increased speed over epithelial cells. Furthermore, the HSH substrate allowed us to physically guide and separate the different cell types based on their relative motile speed. We believe that this method and results will be important in a diversity of areas including mechanical microenvironment, cell motility, and cancer biology.

3D In Vitro Neuron on a Chip for Probing Calcium Mechanostimulation.

The evolution of tissue on a chip systems holds promise for mimicking the response of biological functionality of physiological systems. One important direction for tissue on a chip approaches are neuron-based systems that could mimic neurological responses and lessen the need for in vivo experimentation. For neural research, more attention has been devoted recently to understanding mechanics due to issues in areas such as traumatic brain injury (TBI) and pain, among others. To begin to address these areas, a 3D Nerve Integrated Tissue on a Chip (NITC) approach combined with a Mechanical Excitation Testbed (MET) System is developed to impose external mechanical stimulation toward more realistic physiological environments. PC12 cells differentiated with nerve growth factor, which were cultured in a controlled 3D scaffolds, are used. The cells are labeled in a 3D NITC system with Fluo-4-AM to examine their calcium response under mechanical stimulation synchronized with image capture. Understanding the neural responses to mechanical stimulation beyond 2D systems is very important for neurological studies and future personalized strategies. This work will have implications in a diversity of areas including tissue-on-a-chip systems, biomaterials, and neuromechanics.

Polycarbonate Heat Molding for Soft Lithography.

Soft lithography enables rapid microfabrication of many types of microsystems by replica molding elastomers into master molds. However, master molds can be very costly, hard to fabricate, vulnerable to damage, and have limited casting life. Here, an approach for the multiplication of master molds into monolithic thermoplastic sheets for further soft lithographic fabrication is introduced. The technique is tested with master molds fabricated through photolithography, mechanical micromilling as well as 3D printing, and the results are demonstrated. Microstructures with submicron feature sizes and high aspect ratios are successfully copied. The copying fidelity of the technique is quantitatively characterized and the microfluidic devices fabricated through this technique are functionally tested. This approach is also used to combine different master molds with up to 19 unique geometries into a single monolithic copy mold in a single step displaying the effectiveness of the copying technique over a large footprint area to scale up the microfabrication. This microfabrication technique can be performed outside the cleanroom without using any sophisticated equipment, suggesting a simple way for high-throughput rigid monolithic mold fabrication that can be used in analytical chemistry studies, biomedical research, and microelectromechanical systems.

Chemotactic Responses of Jurkat Cells in Microfluidic Flow-Free Gradient Chambers.

Gradients of soluble molecules coordinate cellular communication in a diverse range of multicellular systems. Chemokine-driven chemotaxis is a key orchestrator of cell movement during organ development, immune response and cancer progression. Chemotaxis assays capable of examining cell responses to different chemokines in the context of various extracellular matrices will be crucial to characterize directed cell motion in conditions which mimic whole tissue conditions. Here, a microfluidic device which can generate different chemokine patterns in flow-free gradient chambers while controlling surface extracellular matrix (ECM) to study chemotaxis either at the population level or at the single cell level with high resolution imaging is presented. The device is produced by combining additive manufacturing (AM) and soft lithography. Generation of concentration gradients in the device were simulated and experimentally validated. Then, stable gradients were applied to modulate chemotaxis and chemokinetic response of Jurkat cells as a model for T lymphocyte motility. Live imaging of the gradient chambers allowed to track and quantify Jurkat cell migration patterns. Using this system, it has been found that the strength of the chemotactic response of Jurkat cells to CXCL12 gradient was reduced by increasing surface fibronectin in a dose-dependent manner. The chemotaxis of the Jurkat cells was also found to be governed not only by the CXCL12 gradient but also by the average CXCL12 concentration. Distinct migratory behaviors in response to chemokine gradients in different contexts may be physiologically relevant for shaping the host immune response and may serve to optimize the targeting and accumulation of immune cells to the inflammation site. Our approach demonstrates the feasibility of using a flow-free gradient chamber for evaluating cross-regulation of cell motility by multiple factors in different biologic processes.

High-throughput mechanotransduction in Drosophila embryos with mesofluidics.

Developing embryos create complexity by expressing genes to coordinate movement which generates mechanical force. An emerging theory is that mechanical force can also serve as an input signal to regulate developmental gene expression. Experimental methods to apply mechanical stimulation to whole embryos have been limited, mainly to aspiration, indentation, or moving a coverslip; these approaches stimulate only a few embryos at a time and require manual alignment. A powerful approach for automation is microfluidic devices, which can precisely manipulate hundreds of samples. However, using microfluidics to apply mechanical stimulation has been limited to small cellular systems, with fewer applications for larger scale whole embryos. We developed a mesofluidic device that applies the precision and automation of microfluidics to the Drosophila embryo: high-throughput automatic alignment, immobilization, compression, real-time imaging, and recovery of hundreds of live embryos. We then use twist:eGFP embryos to show that the mechanical induction of twist depends on the dose and duration of compression. This device allows us to quantify responses to compression, map the distribution of ectopic twist, and measure embryo stiffness. For building mesofluidic devices, we describe modifications on ultra-thick photolithography, derive an analytical model that predicts the deflection of sidewalls, and discuss parametric calibration. This "mesomechanics" approach combines the high-throughput automation and precision of microfluidics with the biological relevance of live embryos to examine mechanotransduction. These analytical models facilitate the design of future devices to process multicellular organisms such as larvae, organoids, and mesoscale tissue samples.

A biosensing soft robot: Autonomous parsing of chemical signals through integrated organic and inorganic interfaces.

The integration of synthetic biology and soft robotics can fundamentally advance sensory, diagnostic, and therapeutic functionality of bioinspired machines. However, such integration is currently impeded by the lack of soft-matter architectures that interface synthetic cells with electronics and actuators for controlled stimulation and response during robotic operation. Here, we synthesized a soft gripper that uses engineered bacteria for detecting chemicals in the environment, a flexible light-emitting diode (LED) circuit for converting biological to electronic signals, and soft pneu-net actuators for converting the electronic signals to movement of the gripper. We show that the hybrid bio-LED-actuator module enabled the gripper to detect chemical signals by applying pressure and releasing the contents of a chemical-infused hydrogel. The biohybrid gripper used chemical sensing and feedback to make actionable decisions during a pick-and-place operation. This work opens previously unidentified avenues in soft materials, synthetic biology, and integrated interfacial robotic systems.

Biomimetic scaffolds with three-dimensional undulated microtopographies.

Many human and animal tissues naturally possess three-dimensional (3D) micro-scale geometries enabling certain physiological functions. Absence of these microgeometries in engineered tissues may undermine the effectiveness of corresponding tissue repair and regeneration. This paper introduces a novel approach to create tissue scaffolds with biomimetic 3D undulated microtopographies. The mechanical micromilling technology is used for precise and reproducible fabrication of poly(methyl methacrylate) (PMMA) master molds with 3D undulated microtopographies. Poly(dimethylsiloxane) (PDMS) production molds are then created using the master molds through elastomer molding. Next, gelatin-chondroitin-6-sulfate-hyaluronic acid (Gel-C6S-HA) is filled into the PDMS molds, lyophilized to obtain solid porous scaffolds, and covalently cross-linked to control biodegradability. The utility of the final porous scaffolds with undulated microtopographies mimicking dermal papillae of skin is demonstrated in vitro by culturing neonatal human fibroblasts (NHFs) on the scaffold surfaces for up to 7 days. The assessment of the mold and scaffold geometries demonstrates high accuracy and reproducibility of the PMMA mold fabrication, as well as well-controlled undulated microtopographies and porous microstructures of the final scaffolds. The analysis of cell responses to the undulated microtopographies shows the biocompatibility and effectiveness of the final scaffolds, as well as unique cellular response to these biomimetic topographies at the macroscopic level.

2D and 3D Mechanobiology in Human and Nonhuman Systems.

Mechanobiology involves the investigation of mechanical forces and their effect on the development, physiology, and pathology of biological systems. The human body has garnered much attention from many groups in the field, as mechanical forces have been shown to influence almost all aspects of human life ranging from breathing to cancer metastasis. Beyond being influential in human systems, mechanical forces have also been shown to impact nonhuman systems such as algae and zebrafish. Studies of nonhuman and human systems at the cellular level have primarily been done in two-dimensional (2D) environments, but most of these systems reside in three-dimensional (3D) environments. Furthermore, outcomes obtained from 3D studies are often quite different than those from 2D studies. We present here an overview of a select group of human and nonhuman systems in 2D and 3D environments. We also highlight mechanobiological approaches and their respective implications for human and nonhuman physiology.

Material Gradients in Stretchable Substrates toward Integrated Electronic Functionality.

The approach toward a stretchable electronic substrate employs multiple soft polymer layers patterned around silicon chips, which act as surrogates for conventional electronics chips, to create a controllable stiffness gradient. Adding just one intermediate polymer layer results in a six-fold increase in the strain failure threshold enabling the substrate to be stretched to over twice its length before delamination occurs.