Microfluidic device for studying cell migration in single or co-existing chemical gradients and electric fields

Cell migration is involved in physiological processes such as wound healing, host defense, and cancer metastasis. The movement of various cell types can be directed by chemical gradients (i.e., chemotaxis). In addition to chemotaxis, many cell types can respond to direct current electric fields (dcEF) by migrating to either the cathode or the anode of the field (i.e., electrotaxis). In tissues, physiological chemical gradients and dcEF can potentially co-exist and the two guiding mechanisms may direct cell migration in a coordinated manner. Recently, microfluidic devices that can precisely configure chemical gradients or dcEF have been increasingly developed and used for chemotaxis and electrotaxis studies. However, a microfluidic device that can configure controlled co-existing chemical gradients and dcEF that would allow quantitative cell migration analysis in complex electrochemical guiding environments is not available. In this study, we developed a polydimethylsiloxane-based microfluidic device that can generate better controlled single or co-existing chemical gradients and dcEF. Using this device, we showed chemotactic migration of T cells toward a chemokine CCL19 gradient or electrotactic migration toward the cathode of an applied dcEF. Furthermore, T cells migrated more strongly toward the cathode of a dcEF in the presence of a competing CCL19 gradient, suggesting the higher electrotactic attraction. Taken together, the developed microfluidic device offers a new experimental tool for studying chemical and electrical guidance for cell migration, and our current results with T cells provide interesting new insights of immune cell migration in complex guiding environments.     

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.     

Microfluidic devices fitted with “flowver” paper pumps generate steady,tunable gradients for extended observation of chemotactic cell migration

Microfluidics approaches have gained popularity in the field of directed cell migration, enabling control of the extracellular environment and integration with live-cell microscopy; however, technical hurdles remain. Among the challenges are the stability and predictability of the environment, which are especially critical for the observation of fibroblasts and other slow-moving cells. Such experiments require several hours and are typically plagued by the introduction of bubbles and other disturbances that naturally arise in standard microfluidics protocols. Here, we report on the development of a passive pumping strategy, driven by the high capillary pressure and evaporative capacity of paper, and its application to study fibroblast chemotaxis. The paper pumps—flowvers (flow + clover)—are inexpensive, compact, and scalable, and they allow nearly bubble-free operation, with a predictable volumetric flow rate on the order of μl/min, for several hours. To demonstrate the utility of this approach, we combined the flowver pumping strategy with a Y-junction microfluidic device to generate a chemoattractant gradient landscape that is both stable (6+ h) and predictable (by finite-element modeling calculations). Integrated with fluorescence microscopy, we were able to recapitulate previous, live-cell imaging studies of fibroblast chemotaxis to platelet derived growth factor (PDGF), with an order-of-magnitude gain in throughput. The increased throughput of single-cell analysis allowed us to more precisely define PDGF gradient conditions conducive for chemotaxis; we were also able to interpret how the orientation of signaling through the phosphoinositide 3-kinase pathway affects the cells’ sensing of and response to conducive gradients.     

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.     

Embryonic stem cell bioprinting for uniform and controlled size embryoid body formation

Embryonic stem cells (ESCs) are pluripotent with multilineage potential to differentiate into virtually all cell types in the organism and thus hold a great promise for cell therapy and regenerative medicine. In vitro differentiation of ESCs starts with a phase known as embryoid body (EB) formation. EB mimics the early stages of embryogenesis and plays an essential role in ESC differentiation in vitro. EB uniformity and size are critical parameters that directly influence the phenotype expression of ESCs. Various methods have been developed to form EBs, which involve natural aggregation of cells. However, challenges persist to form EBs with controlled size, shape, and uniformity in a reproducible manner. The current hanging-drop methods are labor intensive and time consuming. In this study, we report an approach to form controllable, uniform-sized EBs by integrating bioprinting technologies with the existing hanging-drop method. The approach presented here is simple, robust, and rapid. We present significantly enhanced EB size uniformity compared to the conventional manual hanging-drop method.     

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.     

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.     

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 novel microfluidic platform for studying mammalian cell chemotaxis in different oxygen environments under zero-flow conditions

The cell''s micro-environment plays an important role in various physiological and pathological phenomena. To better investigate in vivo cellular behaviors, researchers have expended great effort in building controlled in vitro biophysical and biochemical environments. Because a cell''s gaseous environment affects properties such as its division, metastasis, and differentiation, we developed a zero-flow based platform for studying mammalian cell chemotaxis behavior in different oxygen environments. This platform can construct a linear range of oxygen tensions within one chip (i.e., from 1.4% to 3.6% or 5.5% to 14.5%). To study cell chemotaxis behavior under varying oxygen environments, the chemical gradient direction is established perpendicularly to oxygen change within an observation area. Because the observation area is not subject to flow, shear force is of no concern. In addition, water flow around the cell chambers greatly reduces evaporation and makes long-term microscope imaging possible. In this study, we precisely measure the chemotaxis velocity of MCF-7 human breast cancer cells under different oxygen tension conditions towards CXCL12, which is a stromal cell-derived factor. We find that cell migration rates are not equivalent, even under two close oxygen tensions. We also observed that cells move faster towards high concentrations of chemoattractant when the oxygen tension is below 3% due to the increased expression of HIF-1 (hypoxia-inducible factor 1), which promotes a transition to the amoeboid rather than mesenchymal mode of movement. Our experiments demonstrate that this new microfluidic platform is useful for the quantitative study of mammalian cell chemotaxis under different oxygen conditions in the absence of shear force. We also shed light on the study of chemotaxis under other gaseous environments.     

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…     

An integrated microfluidic system for screening of phage-displayed peptides specific to colon cancer cells and colon cancer stem cells

Affinity reagents recognizing biomarkers specifically are essential components of clinical diagnostics and target therapeutics. However, conventional methods for screening of these reagents often have drawbacks such as large reagent consumption, the labor-intensive or time-consuming procedures, and the involvement of bulky or expensive equipment. Alternatively, microfluidic platforms could potentially automate the screening process within a shorter period of time and reduce reagent and sample consumption dramatically. It has been demonstrated recently that a subpopulation of tumor cells known as cancer stem cells possess high drug resistance and proliferation potential and are regarded as the main cause of metastasis. Therefore, a peptide that recognizes cancer stem cells and differentiates them from other cancer cells will be extremely useful in early diagnosis and target therapy. This study utilized M13 phage display technology to identify peptides that bind, respectively, to colon cancer cells and colon cancer stem cells using an integrated microfluidic system. In addition to positive selection, a negative selection process was integrated on the chip to achieve the selection of peptides of high affinity and specificity. We successfully screened three peptides specific to colon cancer cells and colon cancer stem cells, namely, HOLC-1, HOLC-2, and COLC-1, respectively, and their specificity was measured by the capture rate between target, control, and other cell lines. The capture rates are 43.40 ± 7.23%, 45.16 ± 7.12%, and 49.79 ± 5.34% for colon cancer cells and colon cancer stem cells, respectively, showing a higher specificity on target cells than on control and other cell lines. The developed technique may be promising for early diagnosis of cancer cells and target therapeutics.