Bio-electrospraying and aerodynamically assisted bio-jetting whole human blood: Interrogating cell surface marker integrity

Bio-electrospraying and aerodynamically assisted bio-jetting are two direct cell handling approaches recently pioneered, which have demonstrated significant applicability to the life sciences. These two bioprotocols have undergone scientific rigor, which have seen these techniques been explored in conjunction with a wide range of immortalized, primary and stem cells, and those whole organisms. Those studies have demonstrated a cellular population of >70% viable post-treatment in comparison with controls. Although, these studies assessed cellular viability, cell surface molecules play a critical role in several cellular functions, in particular, have importance to tissue engineering and regenerative medicine. Thus, in the studies reported herein, we demonstrate post-treated viable cells retain their cell surface marker expression levels in comparison to controls, over both short and long time points. Therefore, these studies further push back the frontiers of both bio-electrosprays and aerodynamically assisted bio-jetting in their endeavor as novel strategies for tissue engineering and regenerative biology∕medicine with possible targeted clinical utility.     

The cell's journey: from metaphorical to literal factory

The concept of the cell has been based on metaphor since its inception, and the history of cell theory has continued to rely on metaphor and analogy. In the nineteenth century, cells were most popularly conceived either as building stones or elementary autonomous organisms from which larger organisms are composed. With advances in physiology and the rise of modern biochemistry in the early twentieth century, the chemical factory or laboratory became the dominant metaphor for this biological unit. Today in the twenty-first century, the metaphorical imagery has become a reality, with cells acting as chemical factories for the synthesis of commercially valuable bio-products. The history of the cell shows how metaphors act as conceptual tools, with particular strengths for facilitating different sorts of questions and experimental techniques.     

Aging of cells: accident or programme?

Aging is accompanied by a gradual loss of bodily functions. A wide variety of mechanisms may contribute to the decline in the performance of most organs and tissues. The decrease in several functions can be partially ascribed to a loss of functional cells or, probably more important, an age-related decline in functions of the cells present.Many hypotheses have been proposed to explain cellular aging. It may be genetically programmed, result from damage to the genetic material, be due to errors in the transmission of the genetic information for the synthesis of proteins or reflect changes in cellular proteins after their synthesis. It is doubtful, however, whether a single theory will explain all aspects.     

Assessment of the inhibition of Dengue virus infection by carrageenan via real-time monitoring of cellular oxygen consumption rates within a microfluidic device

A microfluidic device combined with a light modulation system was developed to assess the inhibitory effect of carrageenan on Dengue virus (DENV) infection via real-time monitoring of cellular oxygen consumption rates (OCRs). Measuring cellular OCRs, which can reflect cellular metabolic activity, enabled us to monitor the process of viral infection in real time and to rapidly determine the antiviral activity of potential drugs/chemical compounds. The time variation of the cellular OCR of single cells that were infected in situ by DENV at different multiplicity of infection (m.o.i.) values was first successfully measured within a microfluidic device. The influence of the timing of carrageenan treatment on DENV infection was then examined by real-time monitoring of cellular OCRs in three groups. Cells that were pre-treated with carrageenan and then infected with DENV served as a pre-treatment group, cells to which carrageenan was added simultaneously with DENV served as a virucide group, and cells that were pre-infected with DENV and then treated with carrageenan served as a post-treatment group. By monitoring cellular OCRs, we could rapidly evaluate the inhibitory effect of carrageenan on DENV infection, obtaining a result within 7 h and showing that carrageenan had strong and effective anti-DENV activity in the three groups. In particular, a strong inhibitory effect was observed in the virucide group. Moreover, once the virus enters host cells in the post-treatment group, the immediate treatment with carrageenan for the infected cells has higher efficiency of antiviral activity. Our proposed platform enables to perform time-course or dose-response measurements of changes in cellular metabolic activity caused by diseases, chemical compounds, and drugs via monitoring of the cellular OCR, with rapid and real-time detection. This approach provides the potential to study a wide range of biological applications in cell-based biosensing, toxicology, and drug discovery.     

Apoptosis in health and disease and modulation of apoptosis for therapy: An overview

Apoptosis a physiological mechanism that eliminates excessive, damaged or unwanted cells, is a highly regulated pathway important for maintaining homeostasis in multicellular organisms. It can be initiated through various signals via the extrinsic pathway which involves death receptors, or via the intrinsic pathway which is initiated by intracellular damage and involves the mitochondria and release of cytochrome c from it to further activate caspases. The Bcl-2 family of proteins is situated upstream to the irreversible damage of cellular constituents and is an important checkpoint in the fate of a cell. The pro-apoptotic members, BH3 only members include BID, BAD and BIM. They directly or indirectly activate multidomain BAX/BAK that constitute the requisite gateway to the intrinsic pathway which operates at the mitochondrial surface and endoplasmic reticulum. In contrast, antiapoptotic members such as Bcl-2, Bcl-XL bind and sequester activation. Downstream of mitochondria, the apoptosome involvement is seen to generate caspase activity. Post mitochondria regulation involves IAPs, and their inhibitors. The pathogenesis of several diseases such as cancer, neurodegenerative disorders, autoimmune disorders, heart disease, infectious diseases including AIDS is closely related to aberrant apoptosis. Consequently interest has emerged in employing various the rapeutic approaches such as gene therapy, antisense therapy, recombinant biologicals, organic and combinatorial chemistry, to specifically target apoptosis signaling pathways such as death receptors FAS/TRAIL, Bcl-2, p53, IAPs, SMAC and caspases, etc. and are now advancing from preclinical to clinical phase.     

合成生物学的医学应用研究进展

在医学应用领域,合成生物学以人工设计的基因线路改造人体自身细胞,或改造细菌、病毒等人工生命体,再使其间接作用于人体。这些经人工设计的生命体能够感知疾病特异信号或人工信号、特异性靶向异常细胞和病灶区域、表达报告分子或释放治疗药物,从而实现对人体生理状态的监测,以及对肿瘤、代谢疾病、耐药菌感染等典型疾病的诊断与治疗。文章将综述了合成生物学的医学应用领域近期的一些研究进展。     

pH controlled staining of CD4+ and CD19+ cells within functionalized microfluidic channel

Herein proposed is a simple system to realize hands-free labeling and simultaneous detection of two human cell lines within a microfluidic device. This system was realized by novel covalent immobilization of pH-responsive poly(methacrylic acid) microgels onto the inner glass surface of an assembled polydimethylsiloxane/glass microfluidic channel. Afterwards, selected thiophene labeled monoclonal antibodies, specific for recognition of CD4 antigens on T helper/inducer cells and CD19 antigens on B lymphocytes cell lines, were encapsulated in their active state by the immobilized microgels. When the lymphocytes suspension, containing the two target subpopulations, was flowed through the microchannel, the physiological pH of the cellular suspension induced the release of the labeled antibodies from the microgels and thus the selective cellular staining. The selective pH-triggered staining of the CD4- and CD19-positive cells was investigated in this preliminary experimental study by laser scanning confocal microscopy. This approach represents an interesting and versatile tool to realize cellular staining in a defined module of lab-on-a-chip devices for subsequent detection and counting.     

High-throughput study of alpha-synuclein expression in yeast using microfluidics for control of local cellular microenvironment

Microfluidics is an emerging technology which allows the miniaturization, integration, and automation of fluid handling processes. Microfluidic systems offer low sample consumption, significantly reduced processing time, and the prospect of massive parallelization. A microfluidic platform was developed for the control of the soluble cellular microenvironment of Saccharomyces cerevisiae cells, which enabled high-throughput monitoring of the controlled expression of alpha-synuclein (aSyn), a protein involved in Parkinson's disease. Y-shaped structures were fabricated using particle desorption mass spectrometry-based soft-lithography techniques to generate biomolecular gradients along a microchannel. Cell traps integrated along the microchannel allowed the positioning and monitoring of cells in precise locations, where different, well-controlled chemical environments were established. S. cerevisiae cells genetically engineered to encode the fusion protein aSyn-GFP (green fluorescent protein) under the control of GAL1, a galactose inducible promoter, were loaded in the microfluidic structure. A galactose concentration gradient was established in the channel and a time-dependent aSyn-GFP expression was obtained as a function of the positioning of cells along the galactose gradient. Our results demonstrate the applicability of this microfluidic platform to the spatiotemporal control of cellular microenvironment and open a range of possibilities for the study of cellular processes based on single-cell analysis.     

The use of models and metaphors in developmental biology

That models in science are of a variety of kinds and fulfil a variety of purposes has been particularly evident over the past 30 years in the field of developmental biology. At the beginning of this period, molecular and cellular mechanisms for developmental events were largely lacking, and theoretical models were developed faute de mieux. This gave way to a time in which a number of competing paradigms were present. Now, there is an increasingly detailed knowledge of underlying mechanisms, at a range of levels from genes through cells to organs and organisms. This is therefore a good time to review the usefulness of model making in general, with reference to this discipline.It is suggested that models are of two main kinds: models of structure and models of function. They fulfil three main roles, each of which operates at different stages of maturity of the field of interest. When the causes of some phenomenon are entirely mysterious, then a model may help generate hypotheses for subsequent testing. Second, when competing biological explanations are available, a model may help discriminate between hypotheses. Third, when a well-established hypothesis is available, models may facilitate use of the hypothesis. However, unlike physics, biology is stochastic and contingent, and cannot be entirely deduced from first principles. Mathematical modellers and biologists must remain in constant communication.     

Isolation and manipulation of living adherent cells by micromolded magnetic rafts

A new strategy for magnetically manipulating and isolating adherent cells with extremely high post-collection purity and viability is reported. Micromolded magnetic elements (termed microrafts) were fabricated in an array format and used as culture surfaces and carriers for living, adherent cells. A poly(styrene-co-acrylic acid) polymer containing well dispersed magnetic nanoparticles was developed for creating the microstructures by molding. Nanoparticles of γFe(2)O(3) at concentrations up to 1% wt.∕wt. could be used to fabricate microrafts that were optically transparent, highly magnetic, biocompatible, and minimally fluorescent. To prevent cellular uptake of nanoparticles from the magnetic polymer, a poly(styrene-co-acrylic acid) layer lacking γFe(2)O(3) nanoparticles was placed over the initial magnetic microraft layer to prevent cellular uptake of the γFe(2)O(3) during culture. The microraft surface geometry and physical properties were altered by varying the polymer concentration or layering different polymers during fabrication. Cells plated on the magnetic microrafts were visualized using standard imaging techniques including brightfield, epifluorescence, and confocal microscopy. Magnetic microrafts possessing cells of interest were dislodged from the array and efficiently collected with an external magnet. To demonstrate the feasibility of cell isolation using the magnetic microrafts, a mixed population of wild-type cells and cells stably transfected with a fluorescent protein was plated onto an array. Microrafts possessing single, fluorescent cells were released from the array and magnetically collected. A post-sorting single-cell cloning rate of 92% and a purity of 100% were attained.     

热休克蛋白的研究进展及其应用

在不利的环境中，各种有机体都有其共同对应的分子反应，即正常基因的表达抑制和一组特殊基因－热休克基因的激活和表达，并导致热休克蛋白的大量产生。热休克蛋白主要作为分了伴侣而参与蛋白的折叠、转运及组装等过程，并能恢复或加速清除细胞内已变性的蛋白质而稳定细胞结构，使细胞产生热耐受。随着对热休克蛋白研究的不断深入，其在生物工程、医学和遗传育种等方面的应用前景十分广阔。     

Linear conversion of pressure into concentration, rapid switching of concentration, and generation of linear ramps of concentration in a microfluidic device

Mixing of liquids to produce solutions with different concentrations is one of the basic functionalities of microfluidic devices. Generation of specific temporal patterns of concentration in microfluidic devices is an important technique to study responses of cells and model organisms to variations in the chemical composition of their environment. Here, we present a simple microfluidic network that linearly converts pressure at an inlet into concentration of a soluble reagent in an observation region and also enables independent concurrent linear control of concentrations of two reagents. The microfluidic device has an integrated mixer channel with chaotic three-dimensional flow that facilitates rapid switching of concentrations in a continuous range. A simple pneumatic setup generating linear ramps of pressure is used to produce smooth linear ramps and triangular waves of concentration with different slopes. The use of chaotic vs. laminar mixers is discussed in the context of microfluidic devices providing rapid switching and generating temporal waves of concentration.     

A microfluidic co-culture system to monitor tumor-stromal interactions on a chip

The living cells are arranged in a complex natural environment wherein they interact with extracellular matrix and other neighboring cells. Cell-cell interactions, especially those between distinct phenotypes, have attracted particular interest due to the significant physiological relevance they can reveal for both fundamental and applied biomedical research. To study cell-cell interactions, it is necessary to develop co-culture systems, where different cell types can be cultured within the same confined space. Although the current advancement in lab-on-a-chip technology has allowed the creation of in vitro models to mimic the complexity of in vivo environment, it is still rather challenging to create such co-culture systems for easy control of different colonies of cells. In this paper, we have demonstrated a straightforward method for the development of an on-chip co-culture system. It involves a series of steps to selectively change the surface property for discriminative cell seeding and to induce cellular interaction in a co-culture region. Bone marrow stromal cells (HS5) and a liver tumor cell line (HuH7) have been used to demonstrate this co-culture model. The cell migration and cellular interaction have been analyzed using microscopy and biochemical assays. This co-culture system could be used as a disease model to obtain biological insight of pathological progression, as well as a tool to evaluate the efficacy of different drugs for pharmaceutical studies.     

An electroactive microwell array for trapping and lysing single-bacterial cells

Interest in single-cell analysis has increased because it allows to understand cell metabolism and characterize disease states, cellular adaptation to environmental changes, cell cycles, etc. Here, the authors propose a device to electrically trap and lyse single-bacterial cells in an array format for high-throughput single-cell analysis. The applied electric field is highly deformed and concentrated toward the inside of the microwell structures patterned on the planar electrode. This configuration effectively generates dielectrophoretic force to attract a single cell per well. The microwell has a comparable size to the target bacterial cell making it possible to trap single cells by physically excluding additional cells. Inducing highly concentrated electric potential on the cell membrane can also effectively lyse the trapped single-bacterial cells. The feasibility of the authors' approach was demonstrated by trapping and lysing Escherichia coli cells at the single-cell level. The present microwell array can be used as a basic tool for individual bacterial cell analysis.     

Advances in three-dimensional rapid prototyping of microfluidic devices for biological applications

The capability of 3D printing technologies for direct production of complex 3D structures in a single step has recently attracted an ever increasing interest within the field of microfluidics. Recently, ultrafast lasers have also allowed developing new methods for production of internal microfluidic channels within the bulk of glass and polymer materials by direct internal 3D laser writing. This review critically summarizes the latest advances in the production of microfluidic 3D structures by using 3D printing technologies and direct internal 3D laser writing fabrication methods. Current applications of these rapid prototyped microfluidic platforms in biology will be also discussed. These include imaging of cells and living organisms, electrochemical detection of viruses and neurotransmitters, and studies in drug transport and induced-release of adenosine triphosphate from erythrocytes.     

大麦胚性愈伤组织细胞ODO冷冻断裂面的扫描电镜观察

ODO冷冻断裂法是一项扫描电镜生物样品制备的有效技术,它在生物医学的形态学研究中发挥了重要作用,把这个技术用到植物离体培养物的超微形态研究,是一个新的尝试.大麦胚性愈伤组织用低浓度的戊二醛前固定,再用ODO冷冻断裂法制备扫描电镜样品.本研究主要观察了淀粉粒和淀粉质体在胚性愈伤组织细胞中的分布,因为淀粉粒沉积的变化和质体结构的变化是去分化和再分化过程中的重要特征之一.本文还图示了自制的冷冻断裂器,并对ODO冷冻断裂法的应用作了一些讨论.     

Inducing self-rotation of cells with natural and artificial melanin in a linearly polarized alternating current electric field

The phenomenon of self-rotation observed in naturally and artificially pigmented cells under an applied linearly polarized alternating current (non-rotating) electrical field has been investigated. The repeatable and controllable rotation speeds of the cells were quantified and their dependence on dielectrophoretic parameters such as frequency, voltage, and waveform was studied. Moreover, the rotation behavior of the pigmented cells with different melanin content was compared to quantify the correlation between self-rotation and the presence of melanin. Most importantly, macrophages, which did not originally rotate in the applied non-rotating electric field, began to exhibit self-rotation that was very similar to that of the pigmented cells, after ingesting foreign particles (e.g., synthetic melanin or latex beads). We envision the discovery presented in this paper will enable the development of a rapid, non-intrusive, and automated process to obtain the electrical conductivities and permittivities of cellular membrane and cytoplasm in the near future.     

Microstructured multi-well plate for three-dimensional packed cell seeding and hepatocyte cell culture

In this article, we present a microstructured multi-well plate for enabling three-dimensional (3D) high density seeding and culture of cells through the use of a standard laboratory centrifuge to promote and maintain 3D tissue-like cellular morphology and cell-specific functionality in vitro without the addition of animal derived or synthetic matrices or coagulants. Each well has microfeatures on the bottom that are comprised of a series of ditches/open microchannels. The dimensions of the microchannels promote and maintain 3D tissue-like cellular morphology and cell-specific functionality in vitro. After cell seeding with a standard pipette, the microstructured multi-well plates were centrifuged to tightly pack cells inside the ditches in order to enhance cell-cell interactions and induce formation of 3D cellular structures during cell culture. Cell-cell interactions were optimized based on cell packing by considering dimensions of the ditches/open microchannels, orientation of the microstructured multi-well plate during centrifugation, cell seeding density, and the centrifugal force and time. With the optimized cell packing conditions, we demonstrated that after 7 days of cell culture, primary human hepatocytes adhered tightly together to form cord-like structures that resembled 3D tissue-like cellular architecture. Importantly, cell membrane polarity was restored without the addition of animal derived or synthetic matrices or coagulants.     

Bacterial chemotaxis: molecular biology of a sensory system

All cells respond to chemical stimuli and the nature of the mechanisms involved is thus very important. This article reviews the response of one of the simplest organisms, Escherichia coli, to chemical stimulation, as evidenced by the effect on flagellar movements. Even this elementary sensory-response system involves almost 50 gene products.     

Inverting microwell array chip for the cultivation of human induced pluripotent stem cells with controlled aggregate size and geometrical arrangement

We present a novel cell culture chip, namely, “inverting microwell array chip,” for cultivation of human induced pluripotent stem cells. The chip comprises a lower hydrogel microwell array and an upper polystyrene culture surface. We demonstrate the formation of uniform cellular aggregates in the microwell array, and after inversion, a culture with controlled aggregate size and geometrical arrangement on the polystyrene surface. Here, we report effects of cell concentrations on a cultivation sequence in the chip.