Traumatic brain injuries are the leading cause of disability each year in the US. The most common and devastating consequence is the stretching of axons caused by shear deformation that occurs during rotational acceleration of the brain during injury. The injury effects on axonal molecular and functional events are not fully characterized. We have developed a strain injury model that maintains the three dimensional cell architecture and neuronal networks found in vivo with the ability to visualize individual axons and their response to a mechanical injury. The advantage of this model is that it can apply uniaxial strains to axons that make functional connections between two organotypic slices and injury responses can be observed in real-time and over long term. This uniaxial strain model was designed to be capable of applying an array of mechanical strains at various rates of strain, thus replicating a range of modes of axonal injury. Long term culture, preservation of slice and cell orientation, and slice-slice connection on the device was demonstrated. The device has the ability to strain either individual axons or bundles of axons through the control of microchannel dimensions. The fidelity of the model was verified by observing characteristic responses to various strain injuries which included axonal beading, delayed elastic effects and breakdown in microtubules. Microtubule breakdown was shown to be dependent on the degree of the applied strain field, where maximal breakdown was observed at peak strain and minimal breakdown is observed at low strain. This strain injury model could be a powerful tool in assessing strain injury effects on functional axonal connections.

Traumatic brain injury (TBI) affects 5.3 million people in the United States, and there are 12,500 new cases of spinal cord injury (SCI) every year. There is yet a significant need for in vitro models of TBI and SCI in order to understand the biological mechanisms underlying central nervous system (CNS) injury and to identify and test therapeutics to aid in recovery from neuronal injuries. While TBI or SCI studies have been aided with traditional in vivo and in vitro models, the innate limitations in specificity of injury, isolation of neuronal regions, and reproducibility of these models can decrease their usefulness in examining the neurobiology of injury. Microfluidic devices provide several advantages over traditional methods by allowing researchers to (1) examine the effect of injury on specific neural components, (2) fluidically isolate neuronal regions to examine specific effects on subcellular components, and (3) reproducibly create a variety of injuries to model TBI and SCI. These microfluidic devices are adaptable for modeling a wide range of injuries, and in this review, we will examine different methodologies and models recently utilized to examine neuronal injury. Specifically, we will examine vacuum-assisted axotomy, physical injury, chemical injury, and laser-based axotomy. Finally, we will discuss the benefits and downsides to each type of injury model and discuss how researchers can use these parameters to pick a particular microfluidic device to model CNS injury.

Among the many molecules that contribute to glial scarring, chondroitin sulfate proteoglycans (CSPGs) are known to be potent inhibitors of neuronal regeneration. Chondroitinase ABC (ChABC), a bacterial lyase, degrades the glycosaminoglycan (GAG) side chains of CSPGs and promotes tissue regeneration. However, ChABC is thermally unstable and loses all activity within a few hours at 37 °C under dilute conditions. To overcome this limitation, the discovery of a diverse set of tailor-made random copolymers that complex and stabilize ChABC at physiological temperature is reported. The copolymer designs, which are based on chain length and composition of the copolymers, are identified using an active machine learning paradigm, which involves iterative copolymer synthesis, testing for ChABC thermostability upon copolymer complexation, Gaussian process regression modeling, and Bayesian optimization. Copolymers are synthesized by automated PET-RAFT and thermostability of ChABC is assessed by retained enzyme activity (REA) after 24 h at 37 °C. Significant improvements in REA in three iterations of active learning are demonstrated while identifying exceptionally high-performing copolymers. Most remarkably, one designed copolymer promotes residual ChABC activity near 30%, even after one week and notably outperforms other common stabilization methods for ChABC. Together, these results highlight a promising pathway toward sustained tissue regeneration.

Chronic skin wounds are characterized by poor re-epithelialization, angiogenesis and granulation. Previous work has demonstrated that topical stromal cell-derived growth factor-1 (SDF1) promotes neovascularization, resulting in faster re-epithelialization of skin wounds in diabetic mice. However, the clinical usefulness of such bioactive peptides is limited because they are rapidly degraded in the wound environment due to high levels of proteases. Here, we describe the development of a recombinant fusion protein comprised of SDF1 and an elastin-like peptide that confers the ability to self-assemble into nanoparticles. The fusion protein and recombinant human SDF1 showed similar binding characteristics, as indicated by the measured equilibrium dissociation constant (Kd) for the binding of free SDF1 or the fusion protein to the CXCR4 receptor. The biological activity of SDF1-ELP, as measured by intracellular calcium release in HL60 cells was dose dependent, and also very similar to that of free SDF1. In contrast, the biological activity of SDF1-ELP in vivo was significantly superior to that of free SDF1. When applied to full thickness skin wounds in diabetic mice, wounds treated with SDF1-ELP nanoparticles were 95% closed by day 21, and fully closed by day 28, while wounds treated with free SDF1, ELP alone, or vehicle were only 80% closed by day 21, and took 42 days to fully close. In addition, the SDF1-ELP nanoparticles significantly increased the epidermal and dermal layer of the healed wound, as compared to the other groups. These results indicate SDF1-ELP fusion protein nanoparticles are promising agents for the treatment of chronic skin wounds.

Diffuse axonal injury (DAI) is a devastating consequence of traumatic brain injury, resulting in significant axon and neuronal degeneration. Currently, therapeutic options are limited. Using our brain-on-a-chip device, we evaluated axonal responses to DAI. We observed that axonal diameter plays a significant role in response to strain injury, which correlated to delayed elasticity and inversely correlated to axonal beading and axonal degeneration. When changes in mitochondrial membrane potential (MMP) were monitored an applied strain injury threshold was noted, below which delayed hyperpolarization was observed and above which immediate depolarization occurred. When the NHE-1 inhibitor EIPA was administered before injury, inhibition in both hyperpolarization and depolarization occurred along with axonal degeneration. Therefore, axonal diameter plays a significant role in strain injury and our brain-on-a-chip technology can be used both to understand the biochemical consequences of DAI and screen for potential therapeutic agents.

Autofluorescence of blood has been explored as a label free approach for detection of cell types, as well as for diagnosis and detection of infection, cancer, and other diseases.Although blood autofluorescence is used to indicate the presence of several physiological abnormalities with high sensitivity, it often lacks disease specificity due to use of a limited number of fluorophores in the detection of several abnormal conditions. In addition, the measurement of autofluorescence is sensitive to the type of sample, sample preparation, and spectroscopy method used for the measurement. Therefore, while current blood autofluorescence detection approaches may not be suitable for primary clinical diagnosis, it certainly has tremendous potential in developing methods for large scale screening that can identify high risk groups for further diagnosis using highly specific diagnostic tests. This review discusses the source of blood autofluorescence, the role of spectroscopy methods, and various applications that have used autofluorescence of blood, to explore the potential of blood autofluorescence in biomedical research and clinical applications.

Flow cytometry is an invaluable tool utilized in modern biomedical research and clinical applications requiring high throughput, high resolution particle analysis for cytometric characterization and/or sorting of cells and particles as well as for analyzing results from immunocytometric assays. In recent years, research has focused on developing microfluidic flow cytometers with the motivation of creating smaller, less expensive, simpler, and more autonomous alternatives to conventional flow cytometers. These devices could ideally be highly portable, easy to operate without extensive user training, and utilized for research purposes and/or point-of-care diagnostics especially in limited resource facilities or locations requiring on-site analyses. However, designing a device that fulfills the criteria of high throughput analysis, automation and portability, while not sacrificing performance is not a trivial matter. This review intends to present the current state of the field and provide considerations for further improvement by focusing on the key design components of microfluidic flow cytometers. The recent innovations in particle focusing and detection strategies are detailed and compared. This review outlines performance matrix parameters of flow cytometers that are interdependent with each other, suggesting trade offs in selection based on the requirements of the applications. The ongoing contribution of microfluidics demonstrates that it is a viable technology to advance the current state of flow cytometry and develop automated, easy to operate and cost-effective flow cytometers.

Dendritic cells are the principal antigen presenting cells that are responsible for acquiring and transporting antigen from the peripheral tissue to the secondary lymphoid tissue. There they present it to T cells which ultimately initiate an antigen specific immune response. In vivo, the migration of dendritic cells (DCs) and T cell activation are intimately linked. However, ex vivo systems that facilitate integrated evaluation of DC chemotaxis and resulting T cell activation by migrated DCs are lacking. In this work, we have developed a microfabricated platform that integrates DC chemotaxis with T cell activation. The basic design of the microdevice includes two layers of PDMS, with the top layer comprising the chemotaxis compartment and the bottom layer containing a T cell compartment. In the chemotaxis compartment, the DCs are subjected to a chemokine gradient, and their migratory response is evaluated. In the T cell compartment, rapid DC-induced activation of T cells is evaluated by measuring the level of calcium in T cells. We demonstrate the efficacy of our approach by evaluating the integrated response of mature DCs, whereby the overall T cell activation response is governed both by the chemotaxis and the T cell activation potential of mature DCs relative to immature DCs. Our system provides a powerful platform for systematically probing various aspects of antigen induced immune responses – DC maturation, migration and T cell activation – in an integrated fashion.