Simple Design for Membrane-Free Microphysiological Systems to Model the Blood-Tissue Barriers.

Microphysiological systems (MPS) incorporate physiologically relevant microanatomy, mechanics, and cells to mimic tissue function. Reproducible and standardized in vitro models of tissue barriers, such as the blood-tissue interface (BTI), are critical for next-generation MPS applications in research and industry. Many models of the BTI are limited by the need for semipermeable membranes, use of homogenous cell populations, or 2D culture. These factors limit the relevant endothelial-epithelial contact and 3D transport, which would best mimic the BTI. Current models are also difficult to assemble, requiring precise alignment and layering of components. The work reported herein details the engineering of a BTI-on-a-chip (BTI Chip) that addresses current disadvantages by demonstrating a single layer, membrane-free design. Laminar flow profiles, photocurable hydrogel scaffolds, and human cell lines were used to construct a BTI Chip that juxtaposes an endothelium in direct contact with a 3D engineered tissue. A biomaterial composite, gelatin methacryloyl and 8-arm polyethylene glycol thiol, was used for in situ fabrication of a tissue structure within a Y-shaped microfluidic device. To produce the BTI, a laminar flow profile was achieved by flowing a photocurable precursor solution alongside phosphate buffered saline. Immediately after stopping flow, the scaffold underwent polymerization through a rapid exposure to UV light (<300 mJ·cm-2). After scaffold formation, blood vessel endothelial cells were introduced and allowed to adhere directly to the 3D tissue scaffold, without barriers or phase guides. Fabrication of the BTI Chip was demonstrated in both an epithelial tissue model and blood-brain barrier (BBB) model. In the epithelial model, scaffolds were seeded with human dermal fibroblasts. For the BBB models, scaffolds were seeded with the immortalized glial cell line, SVGP12. The BTI Chip microanatomy was analyzed post facto by immunohistochemistry, showing the uniform production of a patent endothelium juxtaposed with a 3D engineered tissue. Fluorescent tracer molecules were used to characterize the permeability of the BTI Chip. The BTI Chips were challenged with an efflux pump inhibitor, cyclosporine A, to assess physiological function and endothelial cell activation. Operation of physiologically relevant BTI Chips and a novel means for high-throughput MPS generation was demonstrated, enabling future development for drug candidate screening and fundamental biological investigations.

Synthesis of sonicated fibrin nanoparticles that modulate fibrin clot polymerization and enhance angiogenic responses.

Chronic wounds can occur when the healing process is disrupted and the wound remains in a prolonged inflammatory stage that leads to severe tissue damage and poor healing outcomes. Clinically used treatments, such as high density, FDA-approved fibrin sealants, do not provide an optimal environment for native cell proliferation and subsequent tissue regeneration. Therefore, new treatments outside the confines of these conventional fibrin bulk gel therapies are required. We have previously developed flowable, low-density fibrin nanoparticles that, when coupled to keratinocyte growth factor, promote cell migration and epithelial wound closure in vivo. Here, we report a new high throughput method for generating the fibrin nanoparticles using probe sonication, which is less time intensive than the previously reported microfluidic method, and investigate the ability of the sonicated fibrin nanoparticles (SFBN) to promote clot formation and cell migration in vitro. The SFBNs can form a fibrin gel when combined with fibrinogen in the absence of exogenous thrombin, and the polymerization rate and fiber density in these fibrin clots is tunable based on SFBN concentration. Furthermore, fibrin gels made with SFBNs support cell migration in an in vitro angiogenic sprouting assay, which is relevant for wound healing. In this report, we show that SFBNs may be a promising wound healing therapy that can be easily produced and delivered in a flowable formulation.

Rheological Properties of Coordinated Physical Gelation and Chemical Crosslinking in Gelatin Methacryloyl (GelMA) Hydrogels.

Synthetically modified proteins, such as gelatin methacryloyl (GelMA), are growing in popularity for bioprinting and biofabrication. GelMA is a photocurable macromer that can rapidly form hydrogels, while also presenting bioactive peptide sequences for cellular adhesion and proliferation. The mechanical properties of GelMA are highly tunable by modifying the degree of substitution via synthesis conditions, though the effects of source material and thermal gelation have not been comprehensively characterized for lower concentration gels. Herein, the effects of animal source and processing sequence are investigated on scaffold mechanical properties. Hydrogels of 4-6 wt% are characterized. Depending on the temperature at crosslinking, the storage moduli for GelMA derived from pigs, cows, and cold-water fish range from 723 to 7340 Pa, 516 to 3484 Pa, and 294 to 464 Pa, respectively. The maximum storage moduli are achieved only by coordinated physical gelation and chemical crosslinking. In this method, the classic thermo-reversible gelation of gelatin occurs when GelMA is cooled below a thermal transition temperature, which is subsequently "locked in" by chemical crosslinking via photocuring. The effects of coordinated physical gelation and chemical crosslinking are demonstrated by precise photopatterning of cell-laden microstructures, inducing different cellular behavior depending on the selected mechanical properties of GelMA.

Photoinduced reconfiguration to control the protein-binding affinity of azobenzene-cyclized peptides.

The impact of next-generation biorecognition elements (ligands) will be determined by the ability to remotely control their binding activity for a target biomolecule in complex environments. Compared to conventional mechanisms for regulating binding affinity (pH, ionic strength, or chaotropic agents), light provides higher accuracy and rapidity, and is particularly suited for labile targets. In this study, we demonstrate a general method to develop azobenzene-cyclized peptide ligands with light-controlled affinity for target proteins. Light triggers a cis/trans isomerization of the azobenzene, which results in a major structural rearrangement of the cyclic peptide from a non-binding to a binding configuration. Critical to this goal are the ability to achieve efficient photo-isomerization under low light dosage and the temporal stability of both cis and trans isomers. We demonstrated our method by designing photo-switchable peptides targeting vascular cell adhesion marker 1 (VCAM1), a cell marker implicated in stem cell function. Starting from a known VCAM1-binding linear peptide, an ensemble of azobenzene-cyclized variants with selective light-controlled binding were identified by combining in silico design with experimental characterization via spectroscopy and surface plasmon resonance. Variant cycloAZOB[G-VHAKQHRN-K] featured rapid, light-controlled binding of VCAM1 (KD,trans/KD,cis ∼ 130). Biotin-cycloAZOB[G-VHAKQHRN-K] was utilized to label brain microvascular endothelial cells (BMECs), showing co-localization with anti-VCAM1 antibodies in cis configuration and negligible binding in trans configuration.

Chitosan Hydrogels for Synergistic Delivery of Chemotherapeutics to Triple Negative Breast Cancer Cells and Spheroids.

PURPOSE: This study aimed to develop a hydrogel system for treating aggressive triple negative breast cancer (TNBC) via kinetically-controlled delivery of the synergistic drug pair doxorubicin (DOX) and gemcitabine (GEM). A 2D assay was adopted to evaluate therapeutic efficacy by determining combination index (CI), and a 3D assay using cancer spheroids was implemented to assess the potential for translation in vivo. METHODS: The release of DOX and GEM from an acetylated-chitosan (ACS, degree of acetylation χAc = 40 ± 5%) was characterized to identify a combined drug loading that affords release kinetics and dose that are therapeutically synergistic. The selected DOX/GEM-ACS formulation was evaluated in vitro with 2-D and 3-D models of TNBC to determine the combination index (CI) and the tumor volume reduction, respectively. RESULTS: Therapeutically desired release dosages and kinetics of GEM and DOX were achieved. When evaluated with a 2-D model of TNBC, the hydrogel afforded a CI of 0.14, indicating a stronger synergism than concurrent administration of DOX and GEM (CI = 0.23). Finally, the therapeutic hydrogel accomplished a notable volume reduction of the cancer spheroids (up to 30%), whereas the corresponding dosages of free drugs only reduced growth rate. CONCLUSIONS: The ACS hydrogel delivery system accomplishes drug release kinetics and molar ratio that affords strong therapeutically synergism. These results, in combination with the choice of ACS as affordable and highly abundant source material, provide a strong pre-clinical demonstration of the potential of the proposed system for complementing surgical resection of aggressive solid tumors.

Modified gaphene oxide (GO) particles in peptide hydrogels: a hybrid system enabling scheduled delivery of synergistic combinations of chemotherapeutics.

The scheduled delivery of synergistic drug combinations is increasingly recognized as highly effective against advanced solid tumors. Of particular interest are composite systems that release a sequence of drugs with defined kinetics and molar ratios to enhance therapeutic effect, while minimizing the dose to patients. In this work, we developed a homogeneous composite comprising modified graphene oxide (GO) nanoparticles embedded in a Max8 peptide hydrogel, which provides controlled kinetics and molar ratios of release of doxorubicin (DOX) and gemcitabine (GEM). First, modified GO nanoparticles (tGO) were designed to afford high DOX loading and sustained release (18.9% over 72 h and 31.4% over 4 weeks). Molecular dynamics simulations were utilized to model the mechanism of DOX loading as a function of surface modification. In parallel, a Max8 hydrogel was developed to release GEM with faster kinetics and achieve a 10-fold molar ratio to DOX. The selected DOX/tGO nanoparticles were suspended in a GEM/Max8 hydrogel matrix, and the resulting composite was tested against a triple negative breast cancer cell line, MDA-MB-231. Notably, the composite formulation afforded a combination index of 0.093 ± 0.001, indicating a much stronger synergism compared to the DOX-GEM combination co-administered in solution (CI = 0.396 ± 0.034).

In-Line Microelectrode Arrays for Impedance Mapping of Microphysiological Systems.

Herein, a 60-electrode array is fabricated down the length of a microchamber for analysis of a microphysiological system. The electrode array is fabricated by standard photolithographic, metallization, and etching techniques. Permutations of 2-wire impedance measurements (10 Hz to 1 MHz) are made along the length of the microchannel using a multiplexer, Gamry potentiostat, and custom Labview code. An impedance "heat map" is created via custom algorithms. Spatial resolution and mapping capabilities are exhibited using conductive NaCl solutions and 2D cell culture.

Monitoring of Microphysiological Systems: Integrating Sensors and Real-Time Data Analysis toward Autonomous Decision-Making.

Microphysiological systems replicate human organ function and are promising technologies for discovery of translatable biomarkers, pharmaceuticals, and regenerative therapies. Because microphysiological systems require complex microscale anatomical structures and heterogeneous cell populations, a major challenge remains to manufacture and operate these products with reproducible and standardized function. In this Perspective, three stages of microphysiological system monitoring, including process, development, and function, are assessed. The unique features and remaining technical challenges for the required sensors are discussed. Monitoring of microphysiological systems requires nondestructive, continuous biosensors and imaging techniques. With such tools, the extent of cellular and tissue development, as well as function, can be autonomously determined and optimized by correlating physical and chemical sensor outputs with markers of physiological performance. Ultimately, data fusion and analyses across process, development, and function monitors can be implemented to adopt microphysiological systems for broad research and commercial applications.

Platelet-Inspired Nanocells for Targeted Heart Repair After Ischemia/Reperfusion Injury.

Cardiovascular disease is the leading cause of mortality worldwide. While reperfusion therapy is vital for patient survival post-heart attack, it also causes further tissue injury, known as myocardial ischemia/reperfusion (I/R) injury in clinical practice. Exploring ways to attenuate I/R injury is of clinical interest for improving post-ischemic recovery. A platelet-inspired nanocell (PINC) that incorporates both prostaglandin E2 (PGE2)-modified platelet membrane and cardiac stromal cell-secreted factors to target the heart after I/R injury is introduced. By taking advantage of the natural infarct-homing ability of platelet membrane and the overexpression of PGE2 receptors (EPs) in the pathological cardiac microenvironment after I/R injury, the PINCs can achieve targeted delivery of therapeutic payload to the injured heart. Furthermore, a synergistic treatment efficacy can be achieved by PINC, which combines the paracrine mechanism of cell therapy with the PGE2/EP receptor signaling that is involved in the repair and regeneration of multiple tissues. In a mouse model of myocardial I/R injury, intravenous injection of PINCs results in augmented cardiac function and mitigated heart remodeling, which is accompanied by the increase in cycling cardiomyocytes, activation of endogenous stem/progenitor cells, and promotion of angiogenesis. This approach represents a promising therapeutic delivery platform for treating I/R injury.

Integrated phosphorescence-based photonic biosensor (iPOB) for monitoring oxygen levels in 3D cell culture systems.

Physiological processes, such as respiration, circulation, digestion, and many pathologies alter oxygen concentration in the blood and tissue. When designing culture systems to recapitulate the in vivo oxygen environment, it is important to integrate systems for monitoring and controlling oxygen concentration. Herein, we report the design and engineering of a system to remotely monitor and control oxygen concentration inside a device for 3D cell culture. We integrate a photonic oxygen biosensor into the 3D tissue scaffold and regulate oxygen concentration via the control of purging gas flow. The integrated phosphorescence-based oxygen biosensor employs the quenching of palladium-benzoporphyrin by molecular oxygen to transduce the local oxygen concentration in the 3D tissue scaffold. The system is validated by testing the effects of normoxic and hypoxic culture conditions on healthy and tumorigenic breast epithelial cells, MCF-10A cells and BT474 cells, respectively. Under hypoxic conditions, both cell types exhibited upregulation of downstream target genes for the hypoxia marker gene, hypoxia-inducible factor 1α (HIF1A). Lastly, by monitoring the real-time fluctuation of oxygen concentration, we illustrated the formation of hypoxic culture conditions due to limited diffusion of oxygen through 3D tissue scaffolds.

Cardiac Stem Cell Patch Integrated with Microengineered Blood Vessels Promotes Cardiomyocyte Proliferation and Neovascularization after Acute Myocardial Infarction.

Cardiac stem cell (CSC) therapy has shown preclinical and clinical evidence for ischemic heart repair but is limited by low cellular engraftment and survival after transplantation. Previous versions of the cardiac patch strategy improve stem cell engraftment and encourage repair of cardiac tissue. However, cardiac patches that can enhance cardiomyogenesis and angiogenesis at the injured site remain elusive. Therapies that target cardiomyocyte proliferation and new blood vessel formation hold great potential for the protection against acute myocardial infarction (MI). Here, we report a new strategy for creating a vascularized cardiac patch in a facile and modular fashion by leveraging microfluidic hydrodynamic focusing to construct the biomimetic microvessels (BMVs) that include human umbilical vein endothelial cells (HUVECs) lining the luminal surface and then encapsulating the BMVs in a fibrin gel spiked with human CSCs. We show that the endothelialized BMVs mimicked the natural architecture and function of capillaries and that the resultant vascularized cardiac patch (BMV-CSC patch) exhibited equivalent release of paracrine factors compared to those of coculture of genuine human CSCs and HUVECs after 7 days of in vitro culture. In a rat model of acute MI, the BMV-CSC patch therapy induced profound mitotic activities of cardiomyocytes in the peri-infarct region 4 weeks post-treatment. A significant increase in myocardial capillary density was noted in the infarcted hearts that received BMV-CSC patch treatment compared to the infarcted hearts treated with conventional CSC patches. The striking therapeutic benefits and the fast and facile fabrication of the BMV-CSC patch make it promising for practical applications. Our findings suggest that the BMV-CSC patch strategy may open up new possibilities for the treatment of ischemic heart injury.

Neuro-Nano Interfaces: Utilizing Nano-Coatings and Nanoparticles to Enable Next-Generation Electrophysiological Recording, Neural Stimulation, and Biochemical Modulation.

Neural interfaces provide a window into the workings of the nervous system-enabling both biosignal recording and modulation. Traditionally, neural interfaces have been restricted to implanted electrodes to record or modulate electrical activity of the nervous system. Although these electrode systems are both mechanically and operationally robust, they have limited utility due to the resultant macroscale damage from invasive implantation. For this reason, novel nanomaterials are being investigated to enable new strategies to chronically interact with the nervous system at both the cellular and network level. In this feature article, the use of nanomaterials to improve current electrophysiological interfaces, as well as enable new nano-interfaces to modulate neural activity via alternative mechanisms, such as remote transduction of electromagnetic fields are explored. Specifically, this article will review the current use of nanoparticle coatings to enhance electrode function, then an analysis of the cutting-edge, targeted nanoparticle technologies being utilized to interface with both the electrophysiological and biochemical behavior of the nervous system will be provided. Furthermore, an emerging, specialized-use case for neural interfaces will be presented: the modulation of the blood-brain barrier.

Topically applied manganese-porphyrins BMX-001 and BMX-010 display a significant anti-inflammatory response in a mouse model of allergic dermatitis.

In this study, we topically administered two antioxidant compounds, the manganese-porphyrin-derivatives BMX-001 and BMX-010, in a mouse model of allergic dermatitis and compared the efficacy for reduction of itch and inflammation. In vitro effects of BMX-001 and BMX-010 on keratinocytes, bone marrow derived dendritic cells (BMDCs) and T-cells were initially analysed. For assessment of scratching behaviour, BMX-001 and BMX-010 (0.01 and 0.1 %) were topically applied 16 h and/or 1 h before compound 48/80 or toluene-2,4,-diisocyanate (TDI) challenge in a TDI induced mouse dermatitis model. Additionally, assessment of allergic skin inflammation was performed in a similar manner in the TDI model. Post-treatment ear thickness was measured 24 h after TDI challenge and compared to basal values. The mice were sacrificed and the ear auricle was removed for further analysis. In vitro, both BMX substances significantly inhibited cytokine production of keratinocytes as well as of BMDC and T-cell proliferation. Topical treatment with BMX cream resulted in a significant decrease in scratching behaviour in the compound 48/80 model, but not in the TDI model. Mice treated with BMX-001 and BMX-010 showed a moderate dose dependent decrease in ear thickness, and interestingly, the concentration of the cytokines IL-1ß and IL-4 in inflamed skin was reduced by 80-90 % by all treatment options. These first results suggest the potential benefit of a BMX-001 and BMX-010 cream for the treatment of allergic-inflammatory skin diseases.

Exploring inflammatory disease drug effects on neutrophil function.

Neutrophils are critical inflammatory cells; thus, it is important to characterize the effects of drugs on neutrophil function in the context of inflammatory diseases. Herein, chemically guided neutrophil migration, known as chemotaxis, is studied in the context of drug treatment at the single cell level using a microfluidic platform, complemented by cell viability assays and calcium imaging. Three representative drugs known to inhibit surface receptor expression, signaling enzyme activity, and the elevation of intracellular Ca(2+) levels, each playing a significant role in neutrophil chemotactic pathways, are used to examine the in vitro drug effects on cellular behaviors. The microfluidic device establishes a stable concentration gradient of chemokines across a cell culture chamber so that neutrophil migration can be monitored under various drug-exposure conditions. Different time- and concentration-dependent regulatory effects were observed by comparing the motility, polarization, and effectiveness of neutrophil chemotaxis in response to the three drugs. Viability assays revealed distinct drug capabilities in reducing neutrophil viability while calcium imaging clarified the role of Ca(2+) in the neutrophil chemotaxis. This study provides mechanistic insight into the drug effects on neutrophil function, facilitating comparison of current and potential pharmaceutical approaches.

Microfluidics-based in vivo mimetic systems for the study of cellular biology.

The human body is a complex network of molecules, organelles, cells, tissues, and organs: an uncountable number of interactions and transformations interconnect all the system's components. In addition to these biochemical components, biophysical components, such as pressure, flow, and morphology, and the location of all of these interactions play an important role in the human body. Technical difficulties have frequently limited researchers from observing cellular biology as it occurs within the human body, but some state-of-the-art analytical techniques have revealed distinct cellular behaviors that occur only in the context of the interactions. These types of findings have inspired bioanalytical chemists to provide new tools to better understand these cellular behaviors and interactions. What blocks us from understanding critical biological interactions in the human body? Conventional approaches are often too naïve to provide realistic data and in vivo whole animal studies give complex results that may or may not be relevant for humans. Microfluidics offers an opportunity to bridge these two extremes: while these studies will not model the complexity of the in vivo human system, they can control the complexity so researchers can examine critical factors of interest carefully and quantitatively. In addition, the use of human cells, such as cells isolated from donated blood, captures human-relevant data and limits the use of animals in research. In addition, researchers can adapt these systems easily and cost-effectively to a variety of high-end signal transduction mechanisms, facilitating high-throughput studies that are also spatially, temporally, or chemically resolved. These strengths should allow microfluidic platforms to reveal critical parameters in the human body and provide insights that will help with the translation of pharmacological advances to clinical trials. In this Account, we describe selected microfluidic innovations within the last 5 years that focus on modeling both biophysical and biochemical interactions in cellular communication, such as flow and cell-cell networks. We also describe more advanced systems that mimic higher level biological networks, such as organ on-a-chip and animal on-a-chip models. Since the first papers in the early 1990s, interest in the bioanalytical use of microfluidics has grown significantly. Advances in micro-/nanofabrication technology have allowed researchers to produce miniaturized, biocompatible assay platforms suitable for microfluidic studies in biochemistry and chemical biology. Well-designed microfluidic platforms can achieve quick, in vitro analyses on pico- and femtoliter volume samples that are temporally, spatially, and chemically resolved. In addition, controlled cell culture techniques using a microfluidic platform have produced biomimetic systems that allow researchers to replicate and monitor physiological interactions. Pioneering work has successfully created cell-fluid, cell-cell, cell-tissue, tissue-tissue, even organ-like level interfaces. Researchers have monitored cellular behaviors in these biomimetic microfluidic environments, producing validated model systems to understand human pathophysiology and to support the development of new therapeutics.