Squishy Non-Spherical Hydrogel Microparticles

available in PMC 2011 July 15Recent advances in the synthesis of polymeric colloids have opened the doors to new advanced materials. There is strong interest in using these new techniques to produce particles that mimic and/or interact with biological systems. An important characteristic of biological systems that has not yet been exploited in synthetic polymeric colloids is their wide range of deformability. A canonical example of this is the human red blood cell (RBC) which exhibits extreme reversible deformability under flow. Here we report the synthesis of soft polymeric colloids with sizes and shapes that mimic those of the RBC. Additionally, we demonstrate that the mechanical flexibility of the colloids can be reproducibly varied over a large range resulting in RBC-like deformability under physiological flow conditions. These materials have the potential to impact the interaction between biological and synthetic systems.Massachusetts Institute of Technology (MIT-MGH Fellowship in Translational Research)Massachusetts General Hospital (MIT-MGH Fellowship in Translational Research)National Institute of Biomedical Imaging and Bioengineering (U.S.) (BioMEMS Resource Center, P41 EB002503)John Simon Guggenheim Memorial FoundationInstitut Curie (Rothschild-Yvette-Mayent-Institute Curie Fellowship

Nanotechnology: emerging tools for biology and medicine

Historically, biomedical research has been based on two paradigms. First, measurements of biological behaviors have been based on bulk assays that average over large populations. Second, these behaviors have then been crudely perturbed by systemic administration of therapeutic treatments. Nanotechnology has the potential to transform these paradigms by enabling exquisite structures comparable in size with biomolecules as well as unprecedented chemical and physical functionality at small length scales. Here, we review nanotechnology-based approaches for precisely measuring and perturbing living systems. Remarkably, nanotechnology can be used to characterize single molecules or cells at extraordinarily high throughput and deliver therapeutic payloads to specific locations as well as exhibit dynamic biomimetic behavior. These advances enable multimodal interfaces that may yield unexpected insights into systems biology as well as new therapeutic strategies for personalized medicineDamon Runyon Cancer Research Foundation (Merck Fellow, DRG-2065-10)Howard Hughes Medical Institute (Investigator)Lustgarten FoundationNational Institutes of Health (U.S.) (U54CA151884, , Massachusetts Institute of Technology-Harvard Center of Cancer Nanotechnology Excellence)National Institutes of Health (U.S.) (P41- EB002503, BIoMEMS Resource Center

Adaptive-Control Model for Neutrophil Orientation in the Direction of Chemical Gradients

AbstractNeutrophils have a remarkable ability to detect the direction of chemoattractant gradients and move directionally in response to bacterial infections and tissue injuries. For their role in health and disease, neutrophils have been extensively studied, and many of the molecules involved in the signaling mechanisms of gradient detection and chemotaxis have been identified. However, the cellular-scale mechanisms of gradient sensing and directional neutrophil migration have been more elusive, and existent models provide only limited insight into these processes. Here, we propose a what we believe is a novel adaptive-control model for the initiation of cell polarization in response to gradients. In this model, the neutrophils first sample the environment by extending protrusions in random directions and subsequently adapt their sensitivity depending on localized, temporal changes in stimulation levels. Our results suggest that microtubules may play a critical role in integrating all the sensing events from the cellular periphery through their redistribution inside the neutrophils, and may also be involved in modulating local signaling. An unexpected finding was that model neutrophils exhibit significant randomness in timing and directionality of activation, comparable to our experimental observations in microfluidic devices. Moreover, their responses are robust against alterations of the rate and amplitude of the signaling reactions, and for a broad range in chemoattractant concentrations and spatial gradients

MEASUREMENT OF MOLECULAR MOBILITY IN MAMMALIAN CELLS

ABSTRACT Preservation of mammalian cells requires establishing a reversible stasis condition by reducing the intra/extracellular molecular mobility ensuring reduced chemical reaction and deterioration rates. Molecular mobility may be reduced by various techniques. For example, in cryopreservation, mobility within and surrounding the cell is reduced through freezing the free water that constitutes 70-90% of the cell's composition. In dried-state preservation applied successfully to preserve seeds, pharmacological materials and foodstuff (mimicking the anhydrobiosis phenomenon seen in nature), reduction in molecular mobility is established by removing intra/extracellular water. Certain carbohydrates (such as trehalose and sucrose) can be artificially uploaded into mammalian cells to replace the removed water and to form an intra/extracellular glass. In this research, a fluorescent rotor is utilized to determine the changes in intracellular molecular mobility during carbohydrate uploading of mammalian cells. It was shown that using this technique, it is feasible to make real-time mobility measurements at a single cell level

A Raman Microspectroscopy Study of Water and Trehalose in Spin-Dried Cells

AbstractLong-term storage of desiccated nucleated mammalian cells at ambient temperature may be accomplished in a stable glassy state, which can be achieved by removal of water from the biological sample in the presence of glass-forming agents including trehalose. The stability of the glass may be compromised due to a nonuniform distribution of residual water and trehalose within and around the desiccated cells. Thus, quantification of water and trehalose contents at the single-cell level is critical for predicting the glass formation and stability for dry storage. Using Raman microspectroscopy, we estimated the trehalose and residual water contents in the microenvironment of spin-dried cells. Individual cells with or without intracellular trehalose were embedded in a solid thin layer of extracellular trehalose after spin-drying. We found strong evidence suggesting that the residual water was bound at a 2:1 water/trehalose molar ratio in both the extracellular and intracellular milieus. Other than the water associated with trehalose, we did not find any more residual water in the spin-dried sample, intra- or extracellularly. The extracellular trehalose film exhibited characteristics of an amorphous state with a glass transition temperature of ∼22°C. The intracellular milieu also dried to levels suitable for glass formation at room temperature. These findings demonstrate a method for quantification of water and trehalose in desiccated specimens using confocal Raman microspectroscopy. This approach has broad use in desiccation studies to carefully investigate the relationship of water and trehalose content and distribution with the tolerance to drying in mammalian cells

LEA Proteins during Water Stress: Not Just for Plants Anymore

Late embryogenesis abundant (LEA) proteins are extremely hydrophilic proteins that were first identified in land plants. Intracellular accumulation is tightly correlated with acquisition of desiccation tolerance, and data support their capacity to stabilize other proteins and membranes during drying, especially in the presence of sugars like trehalose. Exciting reports now show LEA proteins are not restricted to plants; multiple forms are expressed in desiccation-tolerant animals from at least four phyla. We evaluate here the expression, subcellular localization, biochemical properties and potential functions of LEA proteins in animal species during water stress. LEA proteins are intrinsically unstructured in aqueous solution, but surprisingly, many only assume their native conformation during drying. They are targeted to multiple cellular locations, including mitochondria, and evidence supports that LEA proteins stabilize vitrified sugar glasses thought to be important in the dried state. More in vivo experimentation will be necessary to fully unravel the multiple functional properties of these macromolecules during water stress

Trehalose loading through the mitochondrial permeability transition pore enhances desiccation tolerance in rat liver mitochondria

AbstractTrehalose has extensively been used to improve the desiccation tolerance of mammalian cells. To test whether trehalose improves desiccation tolerance of mammalian mitochondria, we introduced trehalose into the matrix of isolated rat liver mitochondria by reversibly permeabilizing the inner membrane using the mitochondrial permeability transition pore (MPTP). Measurement of the trehalose concentration inside mitochondria using high performance liquid chromatography showed that the sugar permeated rapidly into the matrix upon opening the MPTP. The concentration of intra-matrix trehalose reached 0.29 mmol/mg protein (∼190 mM) in 5 min. Mitochondria, with and without trehalose loaded into the matrix, were desiccated in a buffer containing 0.25 M trehalose by diffusive drying. After re-hydration, the inner membrane integrity was assessed by measurement of mitochondrial membrane potential with the fluorescent probe JC-1. The results showed that following drying to similar water contents, the mitochondria loaded with trehalose had significantly higher inner membrane integrity than those without trehalose loading. These findings suggest the presence of trehalose in the mitochondrial matrix affords improved desiccation tolerance to the isolated mitochondria

Live Pups from Evaporatively Dried Mouse Sperm Stored at Ambient Temperature for up to 2 Years

The purpose of this study is to develop a mouse sperm preservation method based on evaporative drying. Mouse sperm were evaporatively dried and stored at 4°C and ambient temperature for 3 months to 2 years. Upon rehydration, a single sperm was injected into a mature oocyte to develop into a blastocyst after culture or a live birth after embryo transfer to a recipient female. For the samples stored at 4°C for 3, 6, 12, 18, and 24 months, the blastocyst formation rate was 61.5%, 49.1%, 31.5%, 32.2%, and 41.4%, respectively. The blastocyst rate for those stored at ambient temperature (∼22°C) for 3, 6, 12, and 18 months was 57.8%, 36.2%, 33.6%, and 34.4%, respectively. Fifteen, eight and three live pups were produced from sperm stored at room temperature for 12, 18, and 24 months, respectively. This is the first report of live offspring produced from dried mouse sperm stored at ambient temperature for up to 2 years. Based on these results, we suggest that evaporative drying is a potentially useful method for the routine preservation of mouse sperm