Stiffening of Red Blood Cells Induced by Disordered Cytoskeleton Structures: A Joint Theory-experiment Study

The functions and elasticities of the cell are largely related to the structures of the cytoskeletons underlying the lipid bi-layer. Among various cell types, the Red Blood Cell (RBC) possesses a relatively simple cytoskeletal structure. Underneath the membrane, the RBC cytoskeleton takes the form of a two dimensional triangular network, consisting of nodes of actins (and other proteins) and edges of spectrins. Recent experiments focusing on the malaria infected RBCs (iRBCs) showed that there is a correlation between the elongation of spectrins in the cytoskeletal network and the stiffening of the iRBCs. Here we rationalize the correlation between these two observations by combining the worm-like chain (WLC) model for single spectrins and the Effective Medium Theory (EMT) for the network elasticity. We specifically focus on how the disorders in the cytoskeletal network affect its macroscopic elasticity. Analytical and numerical solutions from our model reveal that the stiffness of the membrane increases with increasing end-to-end distances of spectrins, but has a non-monotonic dependence on the variance of the end-to-end distance distributions. These predictions are verified quantitively by our AFM and micropipette aspiration measurements of iRBCs. The model may, from a molecular level, provide guidelines for future identification of new treatment methods for RBC related diseases, such as malaria infection.Comment: 8 pages, 4 figures; 3 supporting figure

Single molecule and multiple bond characterization of catch bond associated cytoadhesion in malaria

The adhesion of malaria infected red blood cells (iRBCs) to host endothelial receptors in the microvasculature, or cytoadhesion, is associated with severe disease pathology such as multiple organ failure and cerebral malaria. Malaria iRBCs have been shown to bind to several receptors, of which intercellular adhesion molecule 1 (ICAM-1) upregulation in brain microvasculature is the only one correlated to cerebral malaria. We utilize a biophysical approach to study the interactions between iRBCs and ICAM-1. At the single molecule level, force spectroscopy experiments reveal that ICAM-1 forms catch bond interactions with Plasmodium falciparum parasite iRBCs. Flow experiments are subsequently conducted to understand multiple bond behavior. Using a robust model that smoothly transitions between our single and multiple bond results, we conclusively demonstrate that the catch bond behavior persists even under flow conditions. The parameters extracted from these experimental results revealed that the rate of association of iRBC-ICAM-1 bonds are ten times lower than iRBC-CD36 (cluster of differentiation 36), a receptor that shows no upregulation in the brains of cerebral malaria patients. Yet, the dissociation rates are nearly the same for both iRBC-receptor interactions. Thus, our results suggest that ICAM-1 may not be the sole mediator responsible for cytoadhesion in the brain

Controlled biomineralization of electrospun poly(ε-caprolactone) fibers for enhancing their mechanical properties

Electrospun polymeric fibers have been investigated as scaffolding materials for bone tissue engineering. However, their mechanical properties, and in particular stiffness and ultimate tensile strength, cannot match those of natural bones. The objective of the study was to develop novel composite nanofiber scaffolds by attaching minerals to polymeric fibers using an adhesive material-the mussel-inspired protein polydopamine-as a superglue . Herein, we report for the first time the use of dopamine to regulate mineralization of electrospun poly(ε-caprolactone) (PCL) fibers to enhance their mechanical properties. We examined the mineralization of the PCL fibers by adjusting the concentration of HCO3 - and dopamine in the mineralized solution, the reaction time and the surface composition of the fibers. We also examined mineralization on the surface of polydopamine-coated PCL fibers. We demonstrated the control of morphology, grain size and thickness of minerals deposited on the surface of electrospun fibers. The obtained mineral coatings render electrospun fibers with much higher stiffness, ultimate tensile strength and toughness, which could be closer to the mechanical properties of natural bone. Such great enhancement of mechanical properties for electrospun fibers through mussel protein-mediated mineralization has not been seen previously. This study could also be extended to the fabrication of other composite materials to better bridge the interfaces between organic and inorganic phases

Viscoelastic Effects of Silicone Gels at the Micro- and Nanoscale

AbstractThere has been an increased use of silicone gel for applications such as cell traction force measurements as well as lab- and organ-on-chip systems. However, silicone gel is a viscoelastic material which tends to undergo non-elastic deformation and displays time-dependent and strain rate-dependent responses. Here, we evaluated the mechanical responses of two types of commonly used silicone gels, Sylgard-184 and CY52-276, when subjected to nanoNewton force and micrometer displacement length scales. Using different mechanical characterization tools and theoretical models, we characterized and quantified the viscoelastic parameters of these substrates. Our experimental results showed that silicone substrates with high stiffness and elasticity and negligible strain rate-dependency and creep responses will be most suited for use at the nanoNewton force and micrometer displacement length scales such as that encountered in cell traction force assays

CD80 and CD86 Differentially Regulate Mechanical Interactions of T-Cells with Antigen-Presenting Dendritic Cells and B-Cells

10.1371/journal.pone.0045185PLoS ONE79

Soft tubular microfluidics for 2D and 3D applications

Microfluidics has been the key component for many applications, including biomedical devices, chemical processors, microactuators, and even wearable devices. This technology relies on soft lithography fabrication which requires cleanroom facilities. Although popular, this method is expensive and labor-intensive. Furthermore, current conventional microfluidic chips precludes reconfiguration, making reiterations in design very time-consuming and costly. To address these intrinsic drawbacks of microfabrication, we present an alternative solution for the rapid prototyping of microfluidic elements such as microtubes, valves, and pumps. In addition, we demonstrate how microtubes with channels of various lengths and cross-sections can be attached modularly into 2D and 3D microfluidic systems for functional applications. We introduce a facile method of fabricating elastomeric microtubes as the basic building blocks for microfluidic devices. These microtubes are transparent, biocompatible, highly deformable, and customizable to various sizes and cross-sectional geometries. By configuring the microtubes into deterministic geometry, we enable rapid, low-cost formation of microfluidic assemblies without compromising their precision and functionality. We demonstrate configurable 2D and 3D microfluidic systems for applications in different domains. These include microparticle sorting, microdroplet generation, biocatalytic micromotor, triboelectric sensor, and even wearable sensing. Our approach, termed soft tubular microfluidics, provides a simple, cheaper, and faster solution for users lacking proficiency and access to cleanroom facilities to design and rapidly construct microfluidic devices for their various applications and needs. Keywords: flexible microfluidics, elastomeric microtubes, microfluidic assemblies, inertial focusing chip, microfluidic sensorSingapore-MIT Alliance for Research and Technology (SMART

Microfluidic label-free selection of mesenchymal stem cell subpopulation during culture expansion extends the chondrogenic potential

Mesenchymal stem cells (MSCs) have been shown as potential candidates for cell-based therapies for a diverse range of tissue regenerative applications. Therapeutic use of MSCs usually requires culture expansion, which increases the heterogeneity of MSCs in vitro, thus affecting the potency of the MSCs for more specific indications. The capacity for identifying and isolating special subsets of MSCs for treatment of specific diseases therefore holds great clinical significance. An important therapeutic application of MSC is for the regeneration of cartilage tissue. We and others have previously developed label-free microfluidic means to isolate subpopulations of culture expanded MSCs based on distinct biophysical characteristics. Here we utilize a spiral micro-channel device to separate culture expanded MSCs into five subgroups according to cell size, and study their proliferation and chondrogenesis at early, middle and late passages. Results show that in all passages, the medium-size subpopulation (cell size of 17-21 μm), compared to other subpopulations, displays significantly higher proliferation rate and chondrogenic capacity in terms of cartilage extracellular matrix formation. Also, the small cell subpopulation (average cell size of 11-12 μm) shows lower viability, and large cell subpopulation (average cell size 23-25 μm) expresses higher level of senescence-associated β-galactosidase. Finally, we show that repeated microfluidic exclusion of MSCs larger than 21 μm and smaller than 17 μm at every passage during continuous culture expansion result in selected MSCs with faster proliferation and better chondrogenic potential as compared to MSC derived from conventional expansion approach. This study demonstrates the significant merit and utility of size-based cell selection for the application of MSCs in cartilage regeneration

Clinical Validation of an Ultra High-Throughput Spiral Microfluidics for the Detection and Enrichment of Viable Circulating Tumor Cells

Background: Circulating tumor cells (CTCs) are cancer cells that can be isolated via liquid biopsy from blood and can be phenotypically and genetically characterized to provide critical information for guiding cancer treatment. Current analysis of CTCs is hindered by the throughput, selectivity and specificity of devices or assays used in CTC detection and isolation. Methodology/Principal Findings: Here, we enriched and characterized putative CTCs from blood samples of patients with both advanced stage metastatic breast and lung cancers using a novel multiplexed spiral microfluidic chip. This system detected putative CTCs under high sensitivity (100%, n = 56) (Breast cancer samples: 12–1275 CTCs/ml; Lung cancer samples: 10–1535 CTCs/ml) rapidly from clinically relevant blood volumes (7.5 ml under 5 min). Blood samples were completely separated into plasma, CTCs and PBMCs components and each fraction were characterized with immunophenotyping (Pan-cytokeratin/CD45, CD44/CD24, EpCAM), fluorescence in-situ hybridization (FISH) (EML4-ALK) or targeted somatic mutation analysis. We used an ultra-sensitive mass spectrometry based system to highlight the presence of an EGFR-activating mutation in both isolated CTCs and plasma cell-free DNA (cf-DNA), and demonstrate concordance with the original tumor-biopsy samples. Conclusions/Significance: We have clinically validated our multiplexed microfluidic chip for the ultra high-throughput, low-cost and label-free enrichment of CTCs. Retrieved cells were unlabeled and viable, enabling potential propagation and real-time downstream analysis using next generation sequencing (NGS) or proteomic analysis.Singapore-MIT Alliance for Research and Technolog

Skin models for cutaneous melioidosis reveal Burkholderia infection dynamics at wound's edge with inflammasome activation, keratinocyte extrusion and epidermal detachment.

Funder: Research Centre of Excellence, Mechanobiology InstituteFunder: National Research FoundationFunder: Cambridge-NUS Global Alliance FundABSTRACTMelioidosis is a serious infectious disease endemic in Southeast Asia, Northern Australia and has been increasingly reported in other tropical and subtropical regions in the world. Percutaneous inoculation through cuts and wounds on the skin is one of the major modes of natural transmission. Despite cuts in skin being a major route of entry, very little is known about how the causative bacterium Burkholderia pseudomallei initiates an infection at the skin and the disease manifestation at the skin known as cutaneous melioidosis. One key issue is the lack of suitable and relevant infection models. Employing an in vitro 2D keratinocyte cell culture, a 3D skin equivalent fibroblast-keratinocyte co-culture and ex vivo organ culture from human skin, we developed infection models utilizing surrogate model organism Burkholderia thailandensis to investigate Burkholderia-skin interactions. Collectively, these models show that the bacterial infection was largely limited at the wound's edge. Infection impedes wound closure, triggers inflammasome activation and cellular extrusion in the keratinocytes as a potential way to control bacterial infectious load at the skin. However, extensive infection over time could result in the epidermal layer being sloughed off, potentially contributing to formation of skin lesions