New mechanism for neural stem cell maintenance in early embryos

Teamning up with co-workers from Japan, UK and US,CAS biochemists have revealed a novel mechanism for maintaining neural stem cells in early embryos. Their work was published on the 6 August issue of Cell Development.     

Microfluidic devices for cell cultivation and proliferation

Microfluidic technology provides precise, controlled-environment, cost-effective, compact, integrated, and high-throughput microsystems that are promising substitutes for conventional biological laboratory methods. In recent years, microfluidic cell culture devices have been used for applications such as tissue engineering, diagnostics, drug screening, immunology, cancer studies, stem cell proliferation and differentiation, and neurite guidance. Microfluidic technology allows dynamic cell culture in microperfusion systems to deliver continuous nutrient supplies for long term cell culture. It offers many opportunities to mimic the cell-cell and cell-extracellular matrix interactions of tissues by creating gradient concentrations of biochemical signals such as growth factors, chemokines, and hormones. Other applications of cell cultivation in microfluidic systems include high resolution cell patterning on a modified substrate with adhesive patterns and the reconstruction of complicated tissue architectures. In this review, recent advances in microfluidic platforms for cell culturing and proliferation, for both simple monolayer (2D) cell seeding processes and 3D configurations as accurate models of in vivo conditions, are examined.     

Microfluidic devices for studying heterotypic cell-cell interactions and tissue specimen cultures under controlled microenvironments

Microfluidic devices allow for precise control of the cellular and noncellular microenvironment at physiologically relevant length- and time-scales. These devices have been shown to mimic the complex in vivo microenvironment better than conventional in vitro assays, and allow real-time monitoring of homotypic or heterotypic cellular interactions. Microfluidic culture platforms enable new assay designs for culturing multiple different cell populations and∕or tissue specimens under controlled user-defined conditions. Applications include fundamental studies of cell population behaviors, high-throughput drug screening, and tissue engineering. In this review, we summarize recent developments in this field along with studies of heterotypic cell-cell interactions and tissue specimen culture in microfluidic devices from our own laboratory.     

Im perfect tools for a difficult job: colposcopy, 'colpocytology' and screening for cervical cancer in Brazil

The quasi-totality of social scientists who studied screening for cervical tumours identified such screening with a single method: the Pap smear (exfoliative cytology). This article explains that this method was not valid everywhere. The history of screening for cervical cancer in Brazil displays an alternative method for detecting cervical malignancies: a direct observation of the cervix with a specific instrument--the colposcope. The development of this method in Brazil in the 1940s and 1950s reflected a complex mixture of professional interests, government policies, and regional, local and charitable initiatives. While the use of colposcopy for cervical tumour screening was phased out in the 1970s and 1980s, the long lifespan and widespread diffusion of this method illuminates the irreducible contingency of specific developments in science, technology and medicine. Seen from the vantage point of Brazil, the Western model for preventing cervical malignancies no longer appears self-evident Alternative choices might have led to the development of different material and visual cultures of medicine, stimulated different patterns of medical specialization and division of medical labour, produced different links between malignancies, women, gynaecologists, epidemiologists and public health experts, and shaped different health policies.     

Manufacturing and wetting low-cost microfluidic cell separation devices

Deterministic lateral displacement (DLD) is a microfluidic size-based particle separation or filter technology with applications in cell separation and enrichment. Currently, there are no cost-effective manufacturing methods for this promising microfluidic technology. In this fabrication paper, however, we develop a simple, yet robust protocol for thermoplastic DLD devices using regulatory-approved materials and biocompatible methods. The final standalone device allowed for volumetric flow rates of 660 μl min⁻¹ while reducing the manufacturing time to <1 h. Optical profilometry and image analysis were employed to assess manufacturing accuracy and precision; the average replicated post height was 0.48% less than the average post height on the master mold and the average replicated array pitch was 1.1% less than the original design with replicated posts heights of 62.1 ± 5.1 μm (mean ± 6 standard deviations) and replicated array pitches of 35.6 ± 0.31 μm.     

New rationale for large metazoan embryo manipulations on chip-based devices

The lack of technologies that combine automated manipulation, sorting, as well as immobilization of single metazoan embryos remains the key obstacle to high-throughput organism-based ecotoxicological analysis and drug screening routines. Noticeably, the major obstacle hampering the automated trapping and arraying of millimetre-sized embryos on chip-based devices is their substantial size and mass, which lead to rapid gravitational-induced sedimentation and strong inertial forces. In this work, we present a comprehensive mechanistic and design rationale for manipulation and passive trapping of individual zebrafish embryos using only hydrodynamic forces. We provide evidence that by employing innovative design features, highly efficient hydrodynamic positioning of large embryos on a chip can be achieved. We also show how computational fluid dynamics-guided design and the Lagrangian particle tracking modeling can be used to optimize the chip performance. Importantly, we show that rapid prototyping and medium scale fabrication of miniaturized devices can be greatly accelerated by combining high-speed laser prototyping with replica moulding in poly(dimethylsiloxane) instead of conventional photolithography techniques. Our work establishes a new paradigm for chip-based manipulation of large multicellular organisms with diameters well above 1 mm and masses often exceeding 1 mg. Passive docking of large embryos is an attractive alternative to provide high level of automation while alleviating potentially deleterious effects associated with the use of active chip actuation. This greatly expands the capabilities of bioanalyses performed on small model organisms and offers numerous and currently inaccessible laboratory automation advantages.     

Optimizing design and fabrication of microfluidic devices for cell cultures: An effective approach to control cell microenvironment in three dimensions

The effects of gradients of bioactive molecules on the cell microenvironment are crucial in several biological processes, such as chemotaxis, angiogenesis, and tumor progression. The elucidation of the basic mechanisms regulating cell responses to gradients requires a tight control of the spatio-temporal features of such gradients. Microfluidics integrating 3D gels are useful tools to fulfill this requirement. However, even tiny flaws in the design or in the fabrication process may severely impair microenvironmental control, thus leading to inconsistent results. Here, we report a sequence of actions aimed at the design and fabrication of a reliable and robust microfluidic device integrated with collagen gel for cell culturing in 3D, subjected to a predetermined gradient of biomolecular signals. In particular, we developed a simple and effective solution to the frequently occurring technical problems of gas bubble formation and 3D matrix collapsing or detaching from the walls. The device here proposed, in Polydimethylsiloxane, was designed to improve the stability of the cell-laden hydrogel, where bubble deprived conditioning media flow laterally to the gel. We report the correct procedure to fill the device with the cell populated gel avoiding the entrapment of gas bubbles, yet maintaining cell viability. Numerical simulations and experiments with fluorescent probes demonstrated the establishment and stability of a concentration gradient across the gel. Finally, chemotaxis experiments of human Mesenchymal Stem Cells under the effects of Bone Morphogenetic Protein-2 gradients were performed in order to demonstrate the efficacy of the system in controlling cell microenvironment. The proposed procedure is sufficiently versatile and simple to be used also for different device geometries or experimental setups.     

Experimental stem cell therapy: biohierarchies and bionetworking in Japan and India

This article concerns new developments in autologous adult stem cell research in Japan and India through the notions of biohierarchy and bionetworking. It conceptualizes how human subject research in one country may be turned into experimental stem cell therapies in another through bionetworks. We analyse the processes that enable researchers in Japan to discard a therapy as being of reputational risk, while researchers in India employ it so that it becomes reputation enhancing. At the same time, scientists from both countries collaborate in and potentially benefit from the same bionetwork. Explaining how the recruitment of patients and scientists is organized through bionetworking, this article analyses how experimental research in India thrives using Japanese technologies. The concept of biohierarchy illustrates how inequalities in health and standards of living in India and in Japan underpin the methods by which researchers, medical professionals, managers and patients collaborate in bionetworks. The concept of 'boundary object' here captures the ways in which the meaning of experimental therapy is defined by subjective categories projected onto it by patients and scientists alike. The article is based on fieldwork conducted by both authors during 3 months between September and December 2008 at various locations in India and Japan. Data for this article were collected from a wide range of interviews with stem cell researchers, medical doctors, coordinators, managers and patients, primary and secondary sources gathered at these centres, and through web and archival research.     

Nanofiber-modified surface directed cell migration and orientation in microsystem

Cell-microscale pattern surface interactions are crucial to understand many fundamental biological questions and develop regenerative medicine and tissue engineering approaches. In this work, we demonstrated a simple method to pattern PDMS surface by sacrificing poly vinyl pyrrolidone (PVP) electrospinning nanofibers and investigated the growth profile of cells on the modified patterned surfaces using stroma cells. The stromal cells were observed to exhibit good viability on this modified surface and the patterned surface with alignment nanofibers could promote cell migration. Furthermore, the modified PDMS surface was integrated with microfluidic channels to create the microscale spatial factor and was used to explore the cell migration and orientation under this microsystem. Both spatial factor and patterned surfaces were found to contribute to the complex cell orientation under the combined dual effects. This established method is simple, fast, and easy for use, demonstrating the potential of this microsystem for applications in addressing biological questions in complex environment.     

Controlled electroporation of the plasma membrane in microfluidic devices for single cell analysis

Chemical cytometry on a single cell level is of interest to various biological fields ranging from cancer to stem cell research. The impact chemical cytometry can exert in these fields depends on the dimensionality of the retrievable analytes content. To this point, the number of different analytes identifiable and additionally their subcellular localization is of interest. To address this, we present an electroporation based approach for selective lysis of only the plasma membrane, which permits analysis of the dissolved cytoplasm, while reducing contributions from the nucleus and membrane bound fractions of the cell analytes. The use of 100 μs long pulse and a well defined DC electric field gradient of ∼4.5 kV·cm⁻¹ generated by 3D electrodes initiates release of a cytoplasm marker in ≪1 s, while retaining nuclear fluorescence markers.     

Microliter-bioreactor array with buoyancy-driven stirring for human hematopoietic stem cell culture

This work presents the development of an array of bioreactors where finely controlled stirring is provided at the microliter scale (100–300 μl). The microliter-bioreactor array is useful for performing protocol optimization in up to 96 parallel experiments of hematopoietic stem cell (HSC) cultures. Exploring a wide range of experimental conditions at the microliter scale minimizes cost and labor. Once the cell culture protocol is optimized, it can be applied to large-scale bioreactors for stem cell production at the clinical level. The controlled stirring inside the wells of a standard 96-well plate is provided by buoyancy-driven thermoconvection. The temperature and velocity fields within the culture volume are determined with numerical simulations. The numerical results are verified with experimental velocity measurements using microparticle image velocimetry (μPIV) and are used to define feasible experimental conditions for stem cell cultures. To test the bioreactor array’s functionality, human umbilical cord blood-derived CD34⁺ cells were cultured for 7 days at five different stirring conditions (0.24–0.58 μm∕s) in six repeated experiments. Cells were characterized in terms of proliferation, and flow cytometry measurements of viability and CD34 expression. The microliter-bioreactor array demonstrates its ability to support HSC cultures under stirred conditions without adversely affecting the cell behavior. Because of the highly controlled operative conditions, it can be used to explore culture conditions where the mass transport of endogenous and exogenous growth factors is selectively enhanced, and cell suspension provided. While the bioreactor array was developed for culturing HSCs, its application can be extended to other cell types.     

A microfluidic cell for studying the formation of regenerated silk by synchrotron radiation small- and wide-angle X-ray scattering

A tube-in-square-pipe microfluidic glass cell has been developed for studying the aggregation and fiber formation from regenerated silk solution by in-situ small-angle X-ray scattering using synchrotron radiation. Acidification-induced aggregation has been observed close to the mixing point of the fibroin and buffer solution. The fibrous, amorphous material is collected in a water bath. Micro-wide-angle X-ray scattering of the dried material confirms its β-sheet nature.     

浅谈成体干细胞及其可塑性

在人类载人宇宙飞船飞向太空、登上月球 ,人类梦想一一实现的今天 ,假如人的各个组织器官能像机器零件那样容易拆卸安装的话 ,我们把已衰竭、恶化的细胞替换掉 ,将新的器官移入体内 ,那么 ,像糖尿病、心脏病等顽疾不复存在 ,各种癌症也能被治愈。这是人类迫切希望实现的又一个梦想。而目前干细胞研究让我们看到了希望。1　干细胞的分类一些学者将干细胞定义为具有自我更新能力的前体细胞 ,它们能生成一种或多种特定的细胞类型。干细胞初步分为胚胎干细胞和成体 (组织 )干细胞两大类。1 .1　胚胎干细胞 ( embryonic stem cell,Esc)是从早期…     

New directive of raloxifene for breast cancer therapy

A survey conducted in 2002 indicates the rising health risks among users of hormone replacement therapy (HRT), an approach for treating menopausal symptoms. Since then, the therapy has been under intense debate about its utility and safety, making patients turn away and search for new treatments.     

Inerter-like devices used for vibration isolation: A historical perspective

Recently, a lot of attention has been given to a mechanical device known as the inerter. It is a mechanical component that can be compared to a capacitor with two ungrounded terminals in the mechanical-electrical systems analogy. In this paper, it is shown that although the concept of an inerter as a separate mechanical element is relatively new, there are several well-established vibration isolation systems that exhibit similar behavior to a simple lumped parameter system containing an inerter. Through a review of the literature, a link is established between the old and new ideas. Furthermore, a comparison between the systems is carried out using the quantities of mechanical impedance and displacement transmissibility. The advantages and disadvantages of using the inerter in vibration isolation are discussed, and a simple way of improving the high-frequency performance without severely degrading the low-frequency performance is described.     

Cloning SU8 silicon masters using epoxy resins to increase feature replicability and production for cell culture devices

In recent years, there has been a dramatic increase in the use of poly(dimethylsiloxane) (PDMS) devices for cell-based studies. Commonly, the negative tone photoresist, SU8, is used to pattern features onto silicon wafers to create masters (SU8-Si) for PDMS replica molding. However, the complexity in the fabrication process, low feature reproducibility (master-to-master variability), silane toxicity, and short life span of these masters have been deterrents for using SU8-Si masters for the production of cell culture based PDMS microfluidic devices. While other techniques have demonstrated the ability to generate multiple devices from a single master, they often do not match the high feature resolution (∼0.1 μm) and low surface roughness that soft lithography masters offer. In this work, we developed a method to fabricate epoxy-based masters that allows for the replication of features with high fidelity directly from SU8-Si masters via their PDMS replicas. By this method, we show that we could obtain many epoxy based masters with equivalent features to a single SU8-Si master with a low feature variance of 1.54%. Favorable feature transfer resolutions were also obtained by using an appropriate Tg epoxy based system to ensure minimal shrinkage of features ranging in size from ∼100 μm to <10 μm in height. We further show that surface coating epoxy masters with Cr/Au lead to effective demolding and yield PDMS chambers that are suitable for long-term culturing of sensitive primary hippocampal neurons. Finally, we incorporated pillars within the Au-epoxy masters to eliminate the process of punching media reservoirs and thereby reducing substantial artefacts and wastage.