Hydrogels for in situ encapsulation of biomimetic membrane arrays

Hydrogels are hydrophilic, porous polymer networks that can absorb up to thousands of times their own weight in water. They have many potential applications, one of which is the encapsulation of freestanding black lipid membranes (BLMs) for novel separation technologies or biosensor applications. We investigated gels for in situ encapsulation of multiple BLMs formed across apertures in a hydrophobic ethylene tetrafluoroethylene (ETFE) support. The encapsulation gels consisted of networks of poly(ethylene glycol)‐dimethacrylate or poly(ethylene glycol)‐diacrylate polymerized using either a chemical initiator or a photoinitiator. The hydrogels were studied with regards to volumetric stability, porosity, and water permeability. All hydrogels had pore sizes around 7 nm with volumetric changes >2% upon crosslinking. Photoinitiated hydrogels had a lower hydraulic water permeability compared to chemically initiated hydrogels; however, for all hydrogels the permeability was several‐fold higher than the water permeability of conventional reverse osmosis (RO) membranes. Lifetimes of freestanding BLM arrays in gel precursor solutions were short compared to arrays formed in buffer. However, polymerizing (crosslinking) the gel stabilized the membranes and resulted in BLM arrays that remained intact for days. This is a substantial improvement over lifetimes for freestanding BLM arrays. Optical images of the membranes and single channel activity of incorporated gramicidin ion channels showed that the lipid membranes retained their integrity and functionality after encapsulation with hydrogel. Our results show that hydrogel encapsulation is a potential means to provide stability for biomimetic devices based on functional proteins reconstituted in biomimetic membrane arrays. Copyright © 2010 John Wiley & Sons, Ltd.     

Drop-based microfluidic devices for encapsulation of single cells

We use microfluidic devices to encapsulate, incubate, and manipulate individual cells in picoliter aqueous drops in a carrier fluid at rates of up to several hundred Hz. We use a modular approach with individual devices for each function, thereby significantly increasing the robustness of our system and making it highly flexible and adaptable to a variety of cell-based assays. The small volumes of the drops enables the concentrations of secreted molecules to rapidly attain detectable levels. We show that single hybridoma cells in 33 pL drops secrete detectable concentrations of antibodies in only 6 h and remain fully viable. These devices hold the promise of developing microfluidic cell cytometers and cell sorters with much greater functionality, allowing assays to be performed on individual cells in their own microenvironment prior to analysis and sorting.     

Truly nonionic polymer shells for the encapsulation of living cells

Engineering surfaces of living cells with natural or synthetic compounds can mediate intercellular communication and provide a protective barrier from hostile agents. We report on truly nonionic hydrogen-bonded LbL coatings for cell surface engineering. These ultrathin, highly permeable polymer membranes are constructed on living cells without the cationic component typically employed to increase the stability of LbL coatings. Without the cytotoxic cationic PEI pre-layer, the viability of encapsulated cells drastically increases to 94%, in contrast to 20% viability in electrostatically-bonded LbL shells. Moreover, the long-term growth of encapsulated cells is not affected, thus facilitating efficient function of protected cells in hostile environment.     

Mussel-inspired encapsulation and functionalization of individual yeast cells

The individual encapsulation of living cells has a great impact on the area of cell-based sensors and devices as well as fundamental studies in cell biology. In this work, living yeast cells were individually encapsulated with functionalizable, artificial polydopamine shells, inspired by an adhesive protein in mussels. Yeast cells maintained their viability within polydopamine, and the cell cycle was controlled by the thickness of the shells. In addition, the artificial shells aided the cell in offering much stronger resistance against foreign aggression, such as lyticase. After formation of the polydopamine shells, the shells were functionalized with streptavidin by utilizing the chemical reactivity of polydopamine, and the functionalized cells were biospecifically immobilized onto the defined surfaces. Our work suggests a biomimetic approach to the encapsulation and functionalization of individual living cells with covalently bonded, artificial shells.     

A microfluidic photolithography for controlled encapsulation of single cells inside hydrogel microstructures

A microfluidic approach to generate hydrogel microstructures inside microchannels for controlled encapsulation of single cells was developed. The method was based on a modified microscope projection photolithography which allowed for the photopolymerization of poly(ethylene glycol) diacrylate (PEG-DA) inside microchannels. Uniform-sized hydrogel microstructures (~50 μm in diameter) were generated one by one with determined positions to encapsulate single cells without losing the viability. Cells of interest could be identified by any kinds of visible labels to be selectively encapsulated inside the formed hydrogel microstructures. Large-scale encapsulation of single cells was achieved with a relatively high efficiency of 80% and the viability of encapsulated cells could be guaranteed by removing the dead cells identified with Trypan blue. This method is simple, fast and convenient to pattern the microchannels with single cells for a wide range of cell-based applications. For demonstration, two intracellular enzyme assays of carboxylesterase were performed to investigate the distribution of enzyme concentrations and the kinetic information within the encapsulated single HepG2 cells.     

Graphene as membrane for encapsulation of yeast cells: protective and electrically conducting

Graphene sheets (chemically reduced), a high modulus and high thermal and electrically conductive material are coupled with yeast cells to form an encapsulating inorganic functional layer. The coupling of the high modulus sheets with the cells increases their stability to osmotic stresses. The sheets also allow the direct visualization of the cells in an electron microscope.     

用于软骨修复的水凝胶*

在软骨组织修复与再生中,水凝胶支架能够为细胞的增殖与分化提供更接近于天然软骨细胞外基质的微环境,是软骨组织修复的一种理想材料。本文介绍了近年发展起来的一些具有新结构和新性能的水凝胶,包括高强度水凝胶以及结构中包含功能蛋白和多肽的水凝胶。重点介绍了对温度、pH值及对生物分子产生响应的刺激响应型水凝胶和自组装水凝胶。具有α-螺旋结构和自组装水凝胶通过两个或多个卷曲螺旋结构聚集形成水凝胶,而通过多肽自组装形成的具有纤维结构的水凝胶在微观的结构上更接近软骨细胞外基质。此外,DNA的分子和序列也用来设计基于DNA的新型水凝胶。本文最后介绍了能在力学、生物学和可注射性等多方面更好地满足软骨修复需要的复合型水凝胶支架、干细胞与水凝胶的复合以及生长因子、基因和一些力学刺激对软骨再生的促进作用。     

Shuttle vectors for studying mutagenesis in mammalian cells

Shuttle vectors are DNA plasmids able to replicate in both mammalian cells and bacteria. They have been used to examine rapidly various aspects of DNA repair, recombination and mutagenesis. Three main classes of shuttle vector have been developed. The transiently replicating vectors are usually based on Simian Virus 40 replication origin. The episomal vectors based on the Epstein-Barr virus replication replicate almost permanently in host cells. Different biological systems, including retroviral vectors, allow the integration of a target gene into the chromosomal structure of the infected cells. In all cases, low molecular weight DNA can be recovered from mammalian cells and shuttled back to bacteria for mutagenesis screening. The advantages and disadvantages of these different types of shuttle vectors are discussed with a special emphasis on their use for a rapid analysis of mutation spectra in mammalian cells.     

Droplet-based microfluidic platforms for the encapsulation and screening of Mammalian cells and multicellular organisms

High-throughput, cell-based assays require small sample volumes to reduce assay costs and to allow for rapid sample manipulation. However, further miniaturization of conventional microtiter plate technology is problematic due to evaporation and capillary action. To overcome these limitations, we describe droplet-based microfluidic platforms in which cells are grown in aqueous microcompartments separated by an inert perfluorocarbon carrier oil. Synthesis of biocompatible surfactants and identification of gas-permeable storage systems allowed human cells, and even a multicellular organism (C. elegans), to survive and proliferate within the microcompartments for several days. Microcompartments containing single cells could be reinjected into a microfluidic device after incubation to measure expression of a reporter gene. This should open the way for high-throughput, cell-based screening that can use >1000-fold smaller assay volumes and has approximately 500x higher throughput than conventional microtiter plate assays.     

Microfabricated devices for biomolecule encapsulation

Biomolecule encapsulation in droplets is important for miniaturizing biological assays to reduce reagent consumption, cost and time of analysis, and can be most effectively achieved by using microfabricated devices. Microfabricated fluidic devices can generate emulsified drops of uniform size with controlled dimensions and contents. Biological and chemical components such as cells, microgels, beads, hydrogel precursors, polymer initiators, and other droplets can be encapsulated within these drops. Encapsulated emulsions are appealing for a variety of applications since drops can be used as tiny reaction vessels to perform high-throughput reactions at fast rates, consuming minimal sample and solvent amounts due to the small size (micron diameters) of the emulsion drops. Facile mixing and droplet coalescence allow for a diversity of assays to be performed on-chip with tunable parameters. The simplicity of operation and speed of analysis with microencapsulated drops lends itself well to an array of quantitative biomolecular studies such as directed evolution, single-molecule DNA amplification, single-cell encapsulation, high-throughput sequencing, enzyme kinetics, and microfluidic cell culture. This review highlights recent advances in the field of microfabricated encapsulating devices, emphasizing the development of emulsifying encapsulations, device design, and current assays that are performed using encapsulating droplets.     

Toward reactant encapsulation for substrate-selectivity

A synthetic tris-(bis-(aminomethyl)pyridine) receptor was prepared in excellent yields via reversible imine condensation strategy. Catalytic activity in Henry reactions of the corresponding copper(II) complex was studied. Capitalizing on previous works by Anslyn with related receptors, the dramatic increase in basicity induced by this type of complex on diketo-derivatives was used to perform a nucleophilic addition of a deprotonated substrate onto an electrophile within the cavity. Hence, a Lewis acid stabilized nitronate was reacted with various aldehydes. A notable preference for small reactants easily accommodated in the cavity over encumbered ones was observed, thus representing an example of substrate-selectivity.     

仿贻贝水凝胶在组织愈合中的应用研究

手术线缝合和订皮钉固定是外科手术中修复组织损伤的常规方法,但是对于相对脆弱的软组织,使用组织粘合剂是代替常规组织修复的重要方法之一.尽管合成的组织粘合剂已经得到广泛应用,但是仍然存在一些缺点,例如湿润环境中粘合性差和潜在毒性等.纤维蛋白胶具有良好的止血性能,但是存在拉伸性和粘附性差、价格昂贵等缺点.仿生粘合剂作为组织粘附剂、止血剂或密封剂在临床手术中应用广泛.然而,在组织创伤的修复应用中,发展耐水粘附、具有生物相容性,多功能一体化的医用粘合剂是近年来研究的热点和难点.自从Messersmith课题组报道了受贻贝启发的多功能聚多巴胺涂层以来,含有酚羟基的材料由于其抗氧化、抗菌消炎等功效,被广泛地应用于医学、食品、化妆品和水处理等领域.仿贻贝水凝胶具有优异的组织粘附性、止血抑菌性、生物安全性和可塑性,是理想的医用粘合剂材料.概述了多酚-合成高分子水凝胶、多酚-生物大分子水凝胶、多酚-无机纳米材料复合水凝胶以及聚多巴胺纳米颗粒复合水凝胶在组织粘附、止血抑菌等方面的研究进展和在组织愈合中的应用探索.总结了多酚水凝胶作为医用组织粘附剂、止血剂、密封剂仍需解决的关键问题,并对此领域的发展趋势进行了...     

组织工程用水凝胶材料

综述了目前用于组织工程支架材料的水凝胶,包括胶原和明胶、透明质酸盐、海藻酸盐,琼脂糖和壳聚糖等天然水凝胶,聚丙烯酸及其衍生物、聚氧化乙烯及其衍生共聚物、聚乙烯醇、聚磷腈和合成多肽等合成水凝胶,并介绍了可注射性组织工程水凝胶。     

微尺度水凝胶与组织工程

水凝胶已被广泛应用于组织工程、药物控释、生物传感器等生物医学领域。随着微制造技术的发展,在纳微米尺度范围内制备形状和尺寸与自然组织相匹配的工程化水凝胶为解决血管化困难、复杂组织结构模拟、多细胞接种等组织工程难题带来了曙光。本文将主要讨论微尺度水凝胶在组织工程领域的研究现状,重点介绍微尺度水凝胶的合成方法及其在血管化和复杂组织结构模拟方面的应用。     

Non-toxic thermotropic liquid crystals for use with mammalian cells

This paper reports the results of a study that aimed to identify thermotropic liquid crystals that are not toxic to mammalian cells. Mesogenic compounds were mixed to create eight liquid crystalline phases, each with a unique set of functional groups. We investigated the toxicity of each liquid crystalline phase using two mammalian cell lines—3T3 fibroblast and SV-40 transformed human corneal epithelial (HCEC) cells. Using dual fluorescent staining assays based on calcein acetoxymethylester (Calcein-AM) and ethidium homodimer, we measured correspondingly the number of viable and dead cells following immersion of the cells in the liquid crystals. It was found that most of the liquid crystals, such as commonly used 5CB and E7, caused cell death after contact with cells for four hours. However, we identified a class of liquid crystals containing fluorophenyl groups that possess minimal or no toxicity (as indicated by results of assays based on Calcein-AM and ethidium homodimer) to cells. Following immersion in fluorophenyl liquid crystals for four hours, the cells were observed to proliferate in culture medium at rates similar to control cells exposed to phosphate-buffered saline (PBS) for four hours. In contrast, treatment of cells for 24 hours with either PBS or liquid crystals (media containing no nutrients and growth factors) resulted in cell death.     

可注射水凝胶及其在再生医学领域的应用

近年来,由工程生物材料制成的可注射治疗剂正变得越来越流行,并推动传统的临床实践走向微创化.可注射水凝胶由于其可调控的物理及化学特性、可控的降解性能、高含水量以及在微创方式下实现递送的能力,在组织工程和药物递送领域中变得越来越重要.研究者们已开发出例如原位交联水凝胶、大孔水凝胶、水凝胶微粒、动态交联水凝胶等一系列性能独特的可注射水凝胶.通过调控水凝胶的固含量和交联密度,并引入适当的共价或非共价相互作用,例如静电相互作用、疏水相互作用等,这些水凝胶可在注射过程中实现生物活性分子的递送.同时,可注射水凝胶亦可用于细胞的递送,提供细胞培养所需的三维环境,并通过调控力学性能、化学修饰、生物功能化修饰等手段调控细胞黏附、增殖、分化等行为.本文旨在回顾近年来可注射水凝胶的设计和制备的相关进展,以及其在再生医学中的应用,并对该领域存在的挑战和潜力进行了展望.     

Methods for rapidly altering the permeability of mammalian cells.

Various agents alter mammalian cells so that they rapidly become nonspecifically permeable to substances that ordinarily do not penetrate intact cells. Thus, toluene renders liver cells permeable to nucleotides and macromolecules. Tween 80 and Tween 60 act on similar fashion, and the effect is reversible. Dextran sulfate reversibly alters the permeability of Ehrlich ascites tumor cells, which offers a tool for studying the control of macromolecular syntheses and other processes. Brief exposure to external ATP alters the permeability of certain transformed mouse cells but not of untransformed cells. The effect of ATP is rapidly reversible.     

Analysis of single mammalian cells on-chip

A goal of modern biology is to understand the molecular mechanisms underlying cellular function. The ability to manipulate and analyze single cells is crucial for this task. The advent of microengineering is providing biologists with unprecedented opportunities for cell handling and investigation on a cell-by-cell basis. For this reason, lab-on-a-chip (LOC) technologies are emerging as the next revolution in tools for biological discovery. In the current discussion, we seek to summarize the state of the art for conventional technologies in use by biologists for the analysis of single, mammalian cells, and then compare LOC devices engineered for these same single-cell studies. While a review of the technical progress is included, a major goal is to present the view point of the practicing biologist and the advances that might increase adoption by these individuals. The LOC field is expanding rapidly, and we have focused on areas of broad interest to the biology community where the technology is sufficiently far advanced to contemplate near-term application in biological experimentation. Focus areas to be covered include flow cytometry, electrophoretic analysis of cell contents, fluorescent-indicator-based analyses, cells as small volume reactors, control of the cellular microenvironment, and single-cell PCR.     

Capillary electrophoresis of single mammalian cells

Capillary electrophoresis (CE) has been established as powerful tool for single cell analysis. Newly developed sampling, separation and detection methods have allowed the investigation of single mammalian cells with CE despite their small size and complex composition. Advances in sample injection techniques include several novel methods for the injection of whole cells and sampling techniques for the study of cellular secretion. CE of single mammalian cells has been applied in a wide range of fields including protein analysis, neuroscience, and oncology. The development of new detection schemes in the analysis of single mammalian cells with CE has included studies of protein expression and the utilization of mass spectrometric and electrochemical detection. Subcellular mammalian cell analysis with CE also has been investigated.