Compact and low-cost deep-ultraviolet microscope system for label-free molecular imaging and point-of-care hematological analysis.

Deep-ultraviolet (UV) microscopy enables label-free, high-resolution, quantitative molecular imaging and enables unique applications in biomedicine, including the potential for fast hematological analysis at the point-of-care. UV microscopy has been shown to quantify hemoglobin content and white blood cells (five-part differential), providing a simple alternative to the current gold standard, the hematological analyzer. Previously, however, the UV system comprised a bulky broadband laser-driven plasma light source along with a large and expensive camera and 3D translation stage. Here, we present a modified deep-UV microscope system with a compact footprint and low-cost components. We detail the novel design with simple, inexpensive optics and hardware to enable fast and accurate automated imaging. We characterize the system, including a modified low-cost web-camera and custom automated 3D translation stage, and demonstrate its ability to scan and capture large area images. We further demonstrate the capability of the system by imaging and analyzing blood smears, using previously trained networks for automatic segmentation, classification (including 5-part white blood cell differential), and colorization. The developed system is approximately 10 times less expensive than previous configurations and can serve as a point-of-care hematology analyzer, as well as be applied broadly in biomedicine as a simple compact, low-cost, quantitative molecular imaging system.

High-Throughput On-Chip Human Mesenchymal Stromal Cell Potency Prediction.

Human mesenchymal stromal cells (hMSCs) are a promising source for regenerative cell therapy. However, hMSC clinical use has been stymied by product variability across hMSC donors and manufacturing practices resulting in inconsistent clinical outcomes. The inability to predict hMSC clinical efficacy, or potency, is a major limitation for market penetration. Standard metrics of hMSC potency employ hMSCs and third-party immune cell co-cultures, however, these assays face translational challenges due to third-party donor variability and lack of scalability. While surrogate markers of hMSC potency have been suggested, none have yet had translational success. To address this, a high-throughput, scalable, low-cost, on-chip microfluidic potency assay is presented with improved functional predictive power and recapitulation of in vivo secretory responses compared to traditional approaches. Comparison of hMSC secretory responses to functional hMSC-medicated immune cell suppression demonstrates shortcomings of current surrogate potency markers and identifies on-chip microfluidic potency markers with improved functional predictive power compared to traditional planar methods. Furthermore, hMSC secretory performance achieved in the on-chip microfluidic system has improved similarity compared to an in vivo model. The results underscore the shortcomings of current culture practices and present a novel system with improved functional predictive power and hMSC physiological responses.

Dosage-dependent antimicrobial activity of DNA-histone microwebs against Staphylococcus aureus.

Neutrophil extracellular traps (NETs) is an antimicrobial cobweb-structured material produced by immune cells for clearance of pathogens in the body, but paradoxically associated with biofilm formation and exacerbated lung infections. To provide a better materials perspective on the pleiotropic roles played by NETs at diverse compositions/concentrations, a NETs-like material (called 'microwebs', abbreviated as µwebs) is synthesized for decoding the antimicrobial activity of NETs against Staphylococcus aureus in infection-relevant conditions. We show that µwebs composed of low-to-intermediate concentrations of DNA-histone complexes successfully trap and inhibit S. aureus growth and biofilm formation. However, with growing concentrations and histone proportions, the resulting microwebs appear gel-like structures accompanied by reduced antimicrobial activity that can even promote formation of S. aureus biofilms. Our simplified model of NETs provides a materials-based evidence on NETs-relevant pathology in the development of biofilms.

Hematocrit significantly confounds diffuse correlation spectroscopy measurements of blood flow.

Diffuse correlation spectroscopy (DCS) is an optical modality used to measure an index of blood flow in biological tissue. This blood flow index depends on both the red blood cell flow rate and density (i.e., hematocrit), although the functional form of hematocrit dependence is not well delineated. Herein, we develop and validate a novel tissue-simulating phantom containing hundreds of microchannels to investigate the influence of hematocrit on blood flow index. For a fixed flow rate, we demonstrate a significant inverse relationship between hematocrit and blood flow index that must be accounted for to accurately estimate blood flow under anemic conditions.

Mechanophenotyping of 3D multicellular clusters using displacement arrays of rendered tractions.

Epithelial tissues mechanically deform the surrounding extracellular matrix during embryonic development, wound repair, and tumor invasion. Ex vivo measurements of such multicellular tractions within three-dimensional (3D) biomaterials could elucidate collective dissemination during disease progression and enable preclinical testing of targeted antimigration therapies. However, past 3D traction measurements have been low throughput due to the challenges of imaging and analyzing information-rich 3D material deformations. Here, we demonstrate a method to profile multicellular clusters in a 96-well-plate format based on spatially heterogeneous contractile, protrusive, and circumferential tractions. As a case study, we profile multicellular clusters across varying states of the epithelial-mesenchymal transition, revealing a successive loss of protrusive and circumferential tractions, as well as the formation of localized contractile tractions with elongated cluster morphologies. These cluster phenotypes were biochemically perturbed by using drugs, biasing toward traction signatures of different epithelial or mesenchymal states. This higher-throughput analysis is promising to systematically interrogate and perturb aberrant mechanobiology, which could be utilized with human-patient samples to guide personalized therapies.

Enabling mesenchymal stromal cell immunomodulatory analysis using scalable platforms.

Human mesenchymal stromal cells (hMSCs) are a promising cell source for numerous regenerative medicine and cell therapy-based applications. However, MSC-based therapies have faced challenges in translation to the clinic, in part due to the lack of sufficient technologies that accurately predict MSC potency and are viable in the context of cell manufacturing. Microfluidic platforms may provide an innovative opportunity to address these challenges by enabling multiparameter analyses of small sample sizes in a high throughput and cost-effective manner, and may provide a more predictive environment in which to analyze hMSC potency. To this end, we demonstrate the feasibility of incorporating 3D culture environments into microfluidic platforms for analysis of hMSC secretory response to inflammatory stimuli and multi-parameter testing using cost-effective and scalable approaches. We first find that the cytokine secretion profile for hMSCs cultured within synthetic poly(ethylene glycol)-based hydrogels is significantly different compared to those cultured on glass substrates, both in growth media and following stimulation with IFN-γ and TNF-α, for cells derived from two donors. For both donors, perfusion with IFN-γ and TNF-α leads to differences in secretion of interleukin 6 (IL-6), interleukin 8 (IL-8), monocyte chemoattractant protein 1 (MCP-1), macrophage colony-stimulating factor (M-CSF), and interleukin-1 receptor antagonist (IL-1ra) between hMSCs cultured in hydrogels and those cultured on glass substrates. We then demonstrate the feasibility of analyzing the response of hMSCs to a stable concentration gradient of soluble factors such as inflammatory stimuli for potential future use in potency analyses, minimizing the amount of sample required for dose-response testing.

Feeling the Force: Measurements of Platelet Contraction and Their Diagnostic Implications.

In addition to the classical biological and biochemical framework, blood clots can also be considered as active biomaterials composed of dynamically contracting platelets, nascent polymeric fibrin that functions as a matrix scaffold, and entrapped blood cells. As platelets sense, rearrange, and apply forces to the surrounding microenvironment, they dramatically change the material properties of the nascent clot, increasing its stiffness by an order of magnitude. Hence, the mechanical properties of blood clots are intricately tied to the forces applied by individual platelets. Research has also shown that the pathophysiological changes in clot mechanical properties are associated with bleeding and clotting disorders, cancer, stroke, ischemic heart disease, and more. By approaching the study of hemostasis and thrombosis from a biophysical and mechanical perspective, important insights have been made into how the mechanics of clotting and the forces applied by platelets are linked to various diseases. This review will familiarize the reader with a mechanics framework that is contextualized with relevant biology. The review also includes a discussion of relevant tools used to study platelet forces either directly or indirectly, and finally, concludes with a summary of potential links between clotting forces and disease.

Morphological single cell profiling of the epithelial-mesenchymal transition.

Single cells respond heterogeneously to biochemical treatments, which can complicate the analysis of in vitro and in vivo experiments. In particular, stressful perturbations may induce the epithelial-mesenchymal transition (EMT), a transformation through which compact, sensitive cells adopt an elongated, resistant phenotype. However, classical biochemical measurements based on population averages over large numbers cannot resolve single cell heterogeneity and plasticity. Here, we use high content imaging of single cell morphology to classify distinct phenotypic subpopulations after EMT. We first characterize a well-defined EMT induction through the master regulator Snail in mammary epithelial cells over 72 h. We find that EMT is associated with increased vimentin area as well as elongation of the nucleus and cytoplasm. These morphological features were integrated into a Gaussian mixture model that classified epithelial and mesenchymal phenotypes with >92% accuracy. We then applied this analysis to heterogeneous populations generated from less controlled EMT-inducing stimuli, including growth factors (TGF-ß1), cell density, and chemotherapeutics (Taxol). Our quantitative, single cell approach has the potential to screen large heterogeneous cell populations for many types of phenotypic variability, and may thus provide a predictive assay for the preclinical assessment of targeted therapeutics.

Wrinkled, wavelength-tunable graphene-based surface topographies for directing cell alignment and morphology.

Textured surfaces with periodic topographical features and long-range order are highly attractive for directing cell-material interactions. They mimic physiological environments more accurately than planar surfaces and can fundamentally alter cell alignment, shape, gene expression, and cellular assembly into superstructures or microtissues. Here we demonstrate for the first time that wrinkled graphene-based surfaces are suitable as textured cell attachment substrates, and that engineered wrinkling can dramatically alter cell alignment and morphology. The wrinkled surfaces are fabricated by graphene oxide wet deposition onto pre-stretched elastomers followed by relaxation and mild thermal treatment to stabilize the films in cell culture medium. Multilayer graphene oxide films form periodic, delaminated buckle textures whose wavelengths and amplitudes can be systematically tuned by variation in the wet deposition process. Human and murine fibroblasts attach to these textured films and remain viable, while developing pronounced alignment and elongation relative to those on planar graphene controls. Compared to lithographic patterning of nanogratings, this method has advantages in the simplicity and scalability of fabrication, as well as the opportunity to couple the use of topographic cues with the unique conductive, adsorptive, or barrier properties of graphene materials for functional biomedical devices.