Tobacco protoplast culture in a polydimethylsiloxane-based microfluidic channel

Summary. Several advances have been made in the use of microfluidic devices for insect and mammalian cell cultures, but no reports of their use for plant cell cultures have been published. We, therefore, conducted a plant cell culture in a microfluidic device using polydimethylsiloxane. Nicotiana tabacum protoplasts were cultured in a variously shaped polydimethylsiloxane channel containing Nitsch medium supplemented with 0.5 g of NLN-13 vitamin mixture, 2.0 mg of α-naphthaleneacetic acid, and 0.5 mg of 6-benzyladenine per liter and 9% mannitol. Protoplasts in the polydimethylsiloxane channel showed cell division and microcolony formation within 4 weeks. The use of a microfluidic channel is a novel technique in the field of plant cell culture. The results of this study will encourage the utilization of polydimethylsiloxane-based microfluidic devices in plant cell engineering and cell analysis. Correspondence and reprints: Department of Biology, Dankook University, 29 San Anseo-dong, Cheonan 300-714, South Korea.     

Low density cell culture of locust neurons in closed-channel microfluidic devices

Microfluidic channel systems were fabricated out of polydimethylsiloxane (PDMS) and used as culture vessels for primary culture of neurons from locust thoracic ganglia. In a biocompatibility study it was shown that cell adhesion and neuronal cell growth of locust neurons on uncoated PDMS was restricted. Coating with concanavalin A improved cell adhesion. In closed-channel microfluidic devices neurons were grown in static-bath culture conditions for more than 15 days. Cell densities of up to 20 cells/channel were not exceeded in low-density cultures but we also found optimal cell growth of single neurons inside individual channels. The first successful cultivation of insect neurons in closed-channel microfluidic devices provides a prerequisite for the development of low density neuronal networks on multi electrode arrays combined with microfluidic devices.     

Review: role of carbon sources for in vitro plant growth and development

In vitro plant cells, tissues and organ cultures are not fully autotrophic establishing a need for carbohydrates in culture media to maintain the osmotic potential, as well as to serve as energy and carbon sources for developmental processes including shoot proliferation, root induction as well as emission, embryogenesis and organogenesis, which are highly energy demanding developmental processes in plant biology. A variety of carbon sources (both reducing and non-reducing) are used in culture media depending upon genotypes and specific stages of growth. However, sucrose is most widely used as a major transport-sugar in the phloem sap of many plants. In micropropagation systems, morphogenetic potential of plant tissues can greatly be manipulated by varying type and concentration of carbon sources. The present article reviews the past and current findings on carbon sources and their sustainable utilization for in vitro plant tissue culture to achieve better growth rate and development.     

New tools and new biology: Recent miniaturized systems for molecular and cellular biology

Recent advances in applied physics and chemistry have led to the development of novel microfluidic systems. Microfluidic systems allow minute amounts of reagents to be processed using μm-scale channels and offer several advantages over conventional analytical devices for use in biological sciences: faster, more accurate and more reproducible analytical performance, reduced cell and reagent consumption, portability, and integration of functional components in a single chip. In this review, we introduce how microfluidics has been applied to biological sciences. We first present an overview of the fabrication of microfluidic systems and describe the distinct technologies available for biological research. We then present examples of microsystems used in biological sciences, focusing on applications in molecular and cellular biology.     

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.     

Quantification of chemical–polymer surface interactions in microfluidic cell culture devices

Microfluidic cell culture devices have been used for drug development, chemical analysis, and environmental pollutant detection. Because of the decreased fluid volume and increased surface area to volume ratio, interactions between device surfaces and the fluid is a key element that affects the performance and detection accuracy of microfluidic devices, particularly if fluid is recirculated by a peristaltic pump. However, this issue has not been studied in detail in a microfluidic cell culture environment. In this study, chemical loss and contaminant leakage from various polymer surfaces in a microfluidic setup were characterized. The effects of hydrophilic coating with Poly (vinyl alcohol), Pluronic® F‐68, and multi‐layer ionic coating were measured. We observed significant surface adsorption of estradiol, doxorubicin, and verapamil with PharMed® BPT tubing, whereas PTFE/BPT and stainless steel/BPT hybrid tubing caused less chemical loss in proportion to the fraction of BPT tubing in the hybrid system. Contaminants leaching out of the BPT tubing were found to be estrogen receptor agonists as determined by estrogen‐induced green fluorescence expression in an estrogen responsive Ishikawa cell line and also caused interference with an estradiol enzyme‐linked immunosorbent assay (ELISA) assay. Stainless steel/BPT hybrid tubing caused the least interference with ELISA. In summary, polymer surface and chemical interactions inside microfluidic systems should not be neglected and require careful investigations when results from a microfluidic system are compared with results from a macroscale cell culture setup. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

iPSC-derived organ-on-a-chip models for personalized human genetics and pharmacogenomics studies

Genome-wide association studies (GWAS) have now correlated hundreds of genetic variants with complex genetic diseases and drug efficacy. Functional characterization of these factors remains challenging, particularly because of the lack of human model systems. Molecular and nanotechnological advances, in particular the ability to generate patient-specific PSC lines, differentiate them into diverse cell types, and seed and combine them on microfluidic chips, have led to the establishment of organ-on-a-chip (OoC) platforms that recapitulate organ biology. OoC technology thus provides unique personalized platforms for studying the effects of host genetics and environmental factors on organ physiology. In this review we describe the technology and provide examples of how OoCs may be used for disease modeling and pharmacogenetic research.     

Frontiers in mammalian cells culture

Summary For the past 60 years, fundamental discoveries in eukaryotic biology using mammalian cell cultures have been significant but modest relative to the enormous potential. Combined with advances in technologies of cell and molecular biology, mammalian cell culture technology is becoming a major, if not essential tool, for fundamental discovery in eukaryotic biology. Reconstruction of the milieu for cells has progressed from simple salt solutions supporting brief survival of tissues outside the body to synthesis of the complete set of structurally defined nutrients, hormones and elements of the extracellular matrix needed to reconstruct complex tissues from cells. The isolation of specific cell types in completely defined environments reveals the true complexity of the mammalian cell and its environment as a dynamic interactive physiological unit. Cell cultures provide the tool for detection and dissection of the mechanism of action of cellular regulators and the genes that determine individual aspects of cell behavior. The technology underpins advances in virology, somatic cell genetics, endocrinology, carcinogenesis, toxicology, pharmacology, hematopoiesis and immunology, and is becoming a major tool in develomental biology, complex tissue physiology and production of unique mammalian cell-derived biologicals in industry. This article is the first of a series of invited reviews aimed at identifying fundamental contributions and current challenges associated with research activities in subdiscriplines of cell and developmental biology in vitro. This treatise is dedicated to Dr. Brian Kimes, Program Director at the National Cancer Institute, whose vision, encouragement and support have contributed significantly to modern developments in mammalian cell culture.     

Fluorescence optical detection in situ for real‐time monitoring of cytochrome P450 enzymatic activity of liver cells in multiple microfluidic devices

We describe an in situ fluorescence optical detection system to demonstrate real‐time and non‐invasive detection of reaction products in a microfluidic device while under perfusion within a standard incubator. The detection system is designed to be compact and robust for operation inside a mammalian cell culture incubator for quantitative detection of fluorescent signal from microfluidic devices. When compared to a standard plate reader, both systems showed similar biphasic response curves with two linear regions. Such a detection system allows real‐time measurements in microfluidic devices with cells without perturbing the culture environment. In a proof‐of‐concept experiment, the cytochrome P450 1A1/1A2 activity of a hepatoma cell line (HepG2/C3A) was monitored by measuring the enzymatic conversion of ethoxyresorufin to resorufin. The hepatoma cell line was embedded in Matrigel^TM construct and cultured in a microfluidic device with medium perfusion. The response of the cells, in terms of P450 1A1/1A2 activity, was significantly different in a plate well system and the microfluidic device. Uninduced cells showed almost no activity in the plate assay, while uninduced cells in Matrigel^TM with perfusion in a microfluidic device showed high activity. Cells in the plate assay showed a significant response to induction with 3‐Methylcholanthrene while cells in the microfluidic device did not respond to the inducer. These results demonstrate that the system is a potentially useful method to measure cell response in a microfluidic system. Biotechnol. Bioeng. 2009; 104: 516–525 © 2009 Wiley Periodicals, Inc.     

Recent advances in single-cell analysis using capillary electrophoresis and microfluidic devices

Cells are the fundamental unit of life, and studies on cell contribute to reveal the mystery of life. However, since variability exists between individual cells even in the same kind of cells, increased emphasis has been put on the analysis of individual cells for getting better understanding on the organism functions. During the past two decades, various techniques have been developed for single-cell analysis. Capillary electrophoresis is an excellent technique for identifying and quantifying the contents of single cells. The microfluidic devices afford a versatile platform for single-cell analysis owing to their unique characteristics. This article provides a review on recent advances in single-cell analysis using capillary electrophoresis and microfluidic devices; focus areas to be covered include sampling techniques, detection methods and main applications in capillary electrophoresis, and cell culture, cell manipulation, chemical cytometry and cellular physiology on microfluidic devices.     

Continuous perfusion microfluidic cell culture array for high-throughput cell-based assays

We present for the first time a microfluidic cell culture array for long-term cellular monitoring. The 10 x 10 array could potentially assay 100 different cell-based experiments in parallel. The device was designed to integrate the processes used in typical cell culture experiments on a single self-contained microfluidic system. Major functions include repeated cell growth/passage cycles, reagent introduction, and real-time optical analysis. The single unit of the array consists of a circular microfluidic chamber, multiple narrow perfusion channels surrounding the main chamber, and four ports for fluidic access. Human carcinoma (HeLa) cells were cultured inside the device with continuous perfusion of medium at 37 degrees C. The observed doubling time was 1.4 +/- 0.1 days with a peak cell density of approximately 2.5*10(5) cells/cm(2). Cell assay was demonstrated by monitoring the fluorescence localization of calcein AM from 1 min to 10 days after reagent introduction. Confluent cell cultures were passaged within the microfluidic chambers using trypsin and successfully regrown, suggesting a stable culture environment suitable for continuous operation. The cell culture array could offer a platform for a wide range of assays with applications in drug screening, bioinformatics, and quantitative cell biology.     

微流控器件-质谱联用接口技术研究进展与应用

生物分析是生命科学研究中的重要环节,分析仪器的小型化是提高生物分析灵敏度、速度、通量和降低成本的有效途径之一.微流控技术能够方便地操纵微量样品,具有集成度高、样品耗量小、污染少等诸多其他常量流控技术难以具备的优点,适用于进行多通道样品处理和高通量分析.除广泛采用的光学和电化学检测手段外,质谱也被用作这些微流控器件的检测器,并逐渐形成了微流控器件-质谱联用技术专门研究领域,进一步促进了自动化程度好、灵敏度高、特异性强的高通量生物分析方法的迅速发展.在大量调研国内外文献的基础上,对微流控器件-质谱联用领域的研究背景和现状进行了综述,不但介绍了微流控器件的制造技术还着重介绍了微流控器件-质谱联用技术在蛋白质组学等生物质谱分析方面的应用和新近进展,评述了可能的发展趋势.     

Sensorineural hearing loss: potential therapies and gene targets for drug development

Recent advances in the developmental biology, genetics and cell biology of the inner ear are guiding research to novel therapeutic modalities - a market currently estimated to be at least US Dollars 10 billion. This article highlights prospects to manipulate the mammalian hearing organ with gene and stem cell delivery to the inner ear to protect, repair or regenerate the hair cells, supporting cells and associated nerves.     

Can we build synthetic,multicellular systems by controlling developmental signaling in space and time?

Using biological machinery to make new, functional molecules is an exciting area in chemical biology. Complex molecules containing both 'natural' and 'unnatural' components are made by processes ranging from enzymatic catalysis to the combination of molecular biology with chemical tools. Here, we discuss applying this approach to the next level of biological complexity -- building synthetic, functional biotic systems by manipulating biological machinery responsible for development of multicellular organisms. We describe recent advances enabling this approach, including first, recent developmental biology progress unraveling the pathways and molecules involved in development and pattern formation; second, emergence of microfluidic tools for delivering stimuli to a developing organism with exceptional control in space and time; third, the development of molecular and synthetic biology toolsets for redesigning or de novo engineering of signaling networks; and fourth, biological systems that are especially amendable to this approach.     

Engineering Microtools in Polymers to Study Cell Biology

Recent advances in surface engineering and soft lithography provide tools to fabricate patterned surfaces and microfluidic devices with dimensions comparable to the sizes of single mammalian cells. These technologies enable the studies of individual cells on spatially well‐defined, patterned surfaces, and in contact with patterned liquid media. They provide information about cells impossible to obtain from traditional biochemical techniques.     

The Golgi apparatus: Lessons from Drosophila

Historically, Drosophila has been a model organism for studying molecular and developmental biology leading to many important discoveries in this field. More recently, the fruit fly has started to be used to address cell biology issues including studies of the secretory pathway, and more specifically on the functional integrity of the Golgi apparatus. A number of advances have been made that are reviewed below. Furthermore, with the development of RNAi technology, Drosophila tissue culture cells have been used to perform genome-wide screens addressing similar issues. Last, the Golgi function has been involved in specific developmental processes, thus shedding new light on the functions of a number of Golgi proteins.     

Constraining the connectivity of neuronal networks cultured on microelectrode arrays with microfluidic techniques: a step towards neuron-based functional chips

In vitro culture of small neuronal networks with pre-defined topological features is particularly desirable when the electrical activity of such assemblies can be monitored for long periods of time. Indeed, it is hoped that such networks, with pre-determined connectivity, will provide unique insights into the structure/function relationship of biological neural networks and their properties of self-organization. However, the experimental techniques that have been developed so far for that purpose have either failed to provide very long-term pattern definition and retention, or they have not shown potential for integration into more complex microfluidic devices. To address this problem, three-dimensional microfluidic systems in poly(dimethylsiloxane) (PDMS) were fabricated and used in conjunction with both custom-made and commercially available planar microelectrode arrays (pMEAs). Various types of primary neuronal cell cultures were established inside these systems. Extracellular electrical signals were successfully recorded from all types of cells placed inside the patterns, and this bioelectrical activity was present for several weeks. The advantage of this approach is that it can be further integrated with microfluidic devices and pMEAs to yield, for example, complex neuron-based biosensors or chips for pharmacological screening.     

Xenopus leads the way: Frogs as a pioneering model to understand the human brain

From its long history in the field of embryology to its recent advances in genetics, Xenopus has been an indispensable model for understanding the human brain. Foundational studies that gave us our first insights into major embryonic patterning events serve as a crucial backdrop for newer avenues of investigation into organogenesis and organ function. The vast array of tools available in Xenopus laevis and Xenopus tropicalis allows interrogation of developmental phenomena at all levels, from the molecular to the behavioral, and the application of CRISPR technology has enabled the investigation of human disorder risk genes in a higher‐throughput manner. As the only major tetrapod model in which all developmental stages are easily manipulated and observed, frogs provide the unique opportunity to study organ development from the earliest stages. All of these features make Xenopus a premier model for studying the development of the brain, a notoriously complex process that demands an understanding of all stages from fertilization to organogenesis and beyond. Importantly, core processes of brain development are conserved between Xenopus and human, underlining the advantages of this model. This review begins by summarizing discoveries made in amphibians that form the cornerstones of vertebrate neurodevelopmental biology and goes on to discuss recent advances that have catapulted our understanding of brain development in Xenopus and in relation to human development and disease. As we engage in a new era of patient‐driven gene discovery, Xenopus offers exceptional potential to uncover conserved biology underlying human brain disorders and move towards rational drug design.     

Cancer Liquid Biopsy Using Integrated Microfluidic Exosome Analysis Platforms

Liquid biopsies serve as both powerful noninvasive diagnostic tools for early cancer screening and prognostic tools for monitoring cancer progression and treatment efficacy. Exosomes are promising biomarkers for liquid biopsies, since these nano‐sized extracellular vesicles (EVs) enrich proteins, lipids, mRNAs, and miRNAs from cells of origin, including cancer cells. Although exosomes are abundantly present in various bodily fluids, conventional exosome isolation and detection methods that rely on benchtop equipment are time‐consuming, expensive, and involve complicated non‐portable procedures. As an alternative, recently developed microfluidic platforms can perform effective exosome separation and detection for liquid biopsies using a single device. Such methods offer advantages of integrity, speed, cost‐efficiency, and portability over conventional benchtop and early microfluidic‐based single‐functional methods which can only separate or detect exosomes separately. These advances have made exosome‐based point‐of‐care (POC) applications possible. This review outlines recent integrated microfluidic‐based exosomal detection strategies to guide future development of such devices for use in liquid biopsies for early cancer screening, prognostic monitoring, and other potential POC applications.     

Microfluidic technology for assisted reproduction.

The physical tools used in assisted reproduction have changed little over several decades. Microfluidics is an emerging technology that allows a fresh examination of the way assisted reproduction is performed. Here we review our work to develop microfluidic devices to perform the functions required in assisted reproduction. These functions include loading/unloading, culture, chemical manipulation, and mechanical manipulation of embryos and oocytes. Basic microfluidic theory and microfluidic device design and operation are discussed. Results are presented for mechanical removal of cumulus cells and for embryo culture. Results suggest that microfluidic systems will lead to improved efficiencies in assisted reproduction.