The MYpop toolbox: Putting yeast stress responses in cellular context on single cell and population scales

Systems biology holds the promise to integrate multiple sources of information in order to build ever more complete models of cellular function. To do this, the field must overcome two significant challenges. First, the current strategy to model average cells must be replaced with population based models accounting for cell‐to‐cell variability. Second, models must be integrated with each other and with basic cellular function. This requires a core model of cellular physiology as well as a multiscale simulation platform to support large‐scale simulation of culture or tissues from single cells. Here, we present such a simulation platform with a core model of yeast physiology as scaffold to integrate and simulate SBML models. The software automates this integration helping users simulate their model of choice in context of the cell division cycle. We benchmark model merging, simulation and analysis by integrating a minimal model of osmotic stress into the core model and analyzing it. We characterize the effect of single cell differences on the dynamics of osmoadaptation, estimating when normal cell growth is resumed and obtaining an explanation for experimentally observed glycerol dynamics based on population dynamics. Hence, the platform can be used to reconcile single cell and population level data.     

Quantification of Tetradesmus obliquus (Chlorophyceae) cell size and lipid content heterogeneity at single‐cell level

Much of our current knowledge of microbial growth is obtained from studies at a population level. Driven by the realization that processes that operate within a population might influence a population's behavior, we sought to better understand Tetradesmus obliquus (formerly Scenedesmus obliquus ) physiology at the cellular level. In this work, an accurate pretreatment method to quantitatively obtain single cells of T. obliquus , a coenobia‐forming alga, is described. These single cells were examined by flow cytometry for triacylglycerol (TAG ), chlorophyll, and protein content, and their cell sizes were recorded by coulter counter. We quantified heterogeneity of size and TAG content at single‐cell level for a population of T. obliquus during a controlled standard batch cultivation. Unexpectedly, variability of TAG content per cell within the population increased throughout the batch run, up to 400 times in the final stage of the batch run, with values ranging from 0.25 to 99 pg · cell^?1. Two subpopulations, classified as having low or high TAG content per cell, were identified. Cell size also increased during batch growth with average values from 36 to 70 μm³ · cell^?1; yet cell size variability increased only up to 16 times. Cell size and cellular TAG content were not correlated at the single‐cell level. Our data show clearly that TAG production is affected by cell‐to‐cell variation, which suggests that its control and better understanding of the underlying processes may improve the productivity of T. obliquus for industrial processes such as biodiesel production.     

Deep Profiling of Cellular Heterogeneity by Emerging Single‐Cell Proteomic Technologies

The ability to comprehensively profile cellular heterogeneity in functional proteome is crucial in advancing the understanding of cell behavior, organism development, and disease mechanisms. Conventional bulk measurement by averaging the biological responses across a population often loses the information of cellular variations. Single‐cell proteomic technologies are becoming increasingly important to understand and discern cellular heterogeneity. The well‐established methods for single‐cell protein analysis based on flow cytometry and fluorescence microscopy are limited by the low multiplexing ability owing to the spectra overlap of fluorophores for labeling antibodies. Recent advances in mass spectrometry (MS), microchip, and reiterative staining‐based techniques for single‐cell proteomics have enabled the evaluation of cellular heterogeneity with high throughput, increased multiplexity, and improved sensitivity. In this review, the principles, developments, advantages, and limitations of these advanced technologies in analysis of single‐cell proteins, along with their biological applications to study cellular heterogeneity, are described. At last, the remaining challenges, possible strategies, and future opportunities that will facilitate the improvement and broad applications of single‐cell proteomic technologies in cell biology and medical research are discussed.     

Coordinated Development of Yeast Colonies: Quantitative Modeling of Diffusion‐Limited Growth – Part 2

A mathematical model was developed which described the growth of yeast colonies based on the assumptions that (i) these populations were built up of single cells whose proliferation was (ii) exclusively controlled by nutrient availability in the environment. The model was of a hybrid cellular automaton type and described discrete cells residing on a one‐dimensional lattice as well as on continuously distributed nutrients. Experimental results and numerical calculations were compared to elucidate under which cultivation conditions the diffusion‐limited growth (DLG) was the major construction principle in yeast colonies. Simulations were scaled to the growth of Yarrowia lipolytica and Candida boidinii colonies under carbon and nitrogen limitation. They showed that nutrient‐controlled growth of the individual cells resulted in DLG of the population. Quantitative predictions for the spatio‐temporal development of the cell‐density profile inside a growing yeast mycelium were compared to the growth characteristics of the model yeast mycelia. Only for the carbon‐limited growth of C. boidinii colonies on glucose as the limiting nutrient resource did the DLG model reproduce the cell‐density profile estimated at the end of the cultivation. Under all other cultivation conditions, strong discrepancies between calculations and experimental results were evident precluding DLG as the ruling regulatory mechanism. Thus, whether or not the development of a yeast population could be described by a DLG scenario, was strongly dependent on the particular cultivation conditions and the applied yeast species. In those cases for which the DLG hypothesis failed to explain the observed growth patterns, the underlying assumptions, i.e., the complete absence of nutrient translocation between the individual cells inside the yeast mycelia as well as the exclusively nutrient‐controlled proliferation of the cells, have to be reevaluated. The presented study demonstrated how the mathematical analysis of growth processes in yeast populations could assist the experimental identification of potential regulatory mechanisms.     

Chemotaxis of cell populations through confined spaces at single-cell resolution

Cell migration is crucial for both physiological and pathological processes. Current in vitro cell motility assays suffer from various drawbacks, including insufficient temporal and/or optical resolution, or the failure to include a controlled chemotactic stimulus. Here, we address these limitations with a migration chamber that utilizes a self-sustaining chemotactic gradient to induce locomotion through confined environments that emulate physiological settings. Dynamic real-time analysis of both population-scale and single-cell movement are achieved at high resolution. Interior surfaces can be functionalized through adsorption of extracellular matrix components, and pharmacological agents can be administered to cells directly, or indirectly through the chemotactic reservoir. Direct comparison of multiple cell types can be achieved in a single enclosed system to compare inherent migratory potentials. Our novel microfluidic design is therefore a powerful tool for the study of cellular chemotaxis, and is suitable for a wide range of biological and biomedical applications.     

Analyzing Microbial Population Heterogeneity—Expanding the Toolbox of Microfluidic Single-Cell Cultivations

Recent research on population heterogeneity revealed fascinating insights into microbial behavior. In particular emerging single-cell technologies, image-based microfluidics lab-on-chip systems generate insights with spatio-temporal resolution, which are inaccessible with conventional tools. This review reports recent developments and applications of microfluidic single-cell cultivation technology, highlighting fields of broad interest such as growth, gene expression and antibiotic resistance and susceptibility. Combining advanced microfluidic single-cell cultivation technology for environmental control with automated time-lapse imaging as well as smart computational image analysis offers tremendous potential for novel investigation at the single-cell level. We propose on-chip control of parameters like temperature, gas supply, pressure or a change in cultivation mode providing a versatile technology platform to mimic more complex and natural habitats. Digital analysis of the acquired images is a requirement for the extraction of biological knowledge and statistically reliable results demand for robust and automated solutions. Focusing on microbial cultivations, we compare prominent software systems that emerged during the last decade, discussing their applicability, opportunities and limitations. Next-generation microfluidic devices with a high degree of environmental control combined with time-lapse imaging and automated image analysis will be highly inspiring and beneficial for fruitful interdisciplinary cooperation between microbiologists and microfluidic engineers and image analysts in the field of microbial single-cell analysis.     

Volumetric stress-strain analysis of optohydrodynamically suspended biological cells

Ongoing investigations are exploring the biomechanical properties of isolated and suspended biological cells in pursuit of understanding single-cell mechanobiology. An optical tweezer with minimal applied laser power has positioned biologic cells at the geometric center of a microfluidic cross-junction, creating a novel optohydrodynamic trap. The resulting fluid flow environment facilitates unique multiaxial loading of single cells with site-specific normal and shear stresses resulting in a physical albeit extensional state. A recent two-dimensional analysis has explored the cytoskeletal strain response due to these fluid-induced stresses [Wilson and Kohles, 2010, "Two-Dimensional Modeling of Nanomechanical Stresses-Strains in Healthy and Diseased Single-Cells During Microfluidic Manipulation," J Nanotechnol Eng Med, 1(2), p. 021005]. Results described a microfluidic environment having controlled nanometer and piconewton resolution. In this present study, computational fluid dynamics combined with multiphysics modeling has further characterized the applied fluid stress environment and the solid cellular strain response in three dimensions to accompany experimental cell stimulation. A volumetric stress-strain analysis was applied to representative living cell biomechanical data. The presented normal and shear stress surface maps will guide future microfluidic experiments as well as provide a framework for characterizing cytoskeletal structure influencing the stress to strain response.     

植物微流芯片——一种实时定量监测生长发育的 高通量整合分析平台

生长发育是一个复杂的动态过程, 了解其发生细节是生命科学研究的重要内容。最新发展起来的微流芯片技术为实现这个目标提供了新的途径。动物及微生物中的应用表明, 该技术兼有实时定量监测和高通量整合处理的优势。在植物研究领域, 用针对根生长特点和要求设计的根微流芯片结合荧光共振能量转移探针已经成功地检测出拟南芥(Arabidopsis thaliana)根细胞内葡萄糖和游离的Ca²⁺、Zn²⁺的浓度。随着各种底物特异的荧光共振能量转移探针的开发和应用, 根微流芯片还可以用来检测植物细胞内激素或其它代谢中间产物的浓度及其动态变化过程。不仅如此, 以微流芯片为基础发展起来的Plant Chip和Tip Chip则为研究植物与微生物的相互作用以及植物花粉管极性生长和细胞分裂分化提供了理想的平台。作为了解遗传因素或环境刺激导致细胞生命活动变化细节的有力工具, 微流芯片技术有望为植物研究领域带来更多新的进展和突破。     

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.     

A part‐dependent account of biological individuality: why holobionts are individuals and ecosystems simultaneously

Given one conception of biological individuality (evolutionary, physiological, etc.), can a holobiont – that is the host + its symbiotic (mutualistic, commensalist and parasitic) microbiome – be simultaneously a biological individual and an ecological community? Herein, we support this possibility by arguing that the notion of biological individuality is part‐dependent. In our account, the individuality of a biological ensemble should not only be determined by the conception of biological individuality in use, but also by the biological characteristics of the part of the ensemble under investigation. In the specific case of holobionts, evaluations of their individuality should be made either host‐relative or microbe‐relative. We support the claim that biological individuality is part‐dependent by drawing upon recent empirical evidence regarding the physiology of hosts and microbes, and the recent characterization of the ‘demibiont’. Our account shows that contemporary disagreements about the individuality of the holobiont derive from an incorrect understanding of the ontology of biological individuality. We show that collaboration between philosophers and biologists can be very fruitful in attempts to solve some contemporary biological debates.     

Microfluidic device‐assisted etching of p‐HEMA for cell or protein patterning

The construction of biomaterials with which to limit the growth of cells or to limit the adsorption of proteins is essential for understanding biological phenomena. Here, we describe a novel method to simply and easily create thin layers of poly (2‐hydroxyethyl methacrylate) (p‐HEMA) for protein and cellular patterning via etching with ethanol and microfluidic devices. First, a cell culture surface or glass coverslip is coated with p‐HEMA. Next, a polydimethylsiloxane (PDMS) microfluidic is placed onto the p‐HEMA surface, and ethanol is aspirated through the device. The PDMS device is removed, and the p‐HEMA surface is ready for protein adsorption or cell plating. This method allows for the fabrication of 0.3 µm thin layers of p‐HEMA, which can be etched to 10 µm wide channels. Furthermore, it creates regions of differential protein adhesion, as shown by Coomassie staining and fluorescent labeling, and cell adhesion, as demonstrated by C2C12 myoblast growth. This method is simple, versatile, and allows biologists and bioengineers to manipulate regions for cell culture adhesion and growth. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:243–248, 2018     

Synthesizing artificial cells from giant unilamellar vesicles: State‐of‐the art in the development of microfluidic technology

Microfluidic technology – the manipulation of fluids at micrometer scales – has revolutionized many areas of synthetic biology. The bottom‐up synthesis of “minimal” cell models has traditionally suffered from poor control of assembly conditions. Giant unilamellar vesicles (GUVs) are good models of living cells on account of their size and unilamellar membrane structure. In recent years, a number of microfluidic approaches for constructing GUVs has emerged. These provide control over traditionally elusive parameters of vesicular structure, such as size, lamellarity, membrane composition, and internal contents. They also address sophisticated cellular functions such as division and protein synthesis. Microfluidic techniques for GUV synthesis can broadly be categorized as continuous‐flow based approaches and droplet‐based approaches. This review presents the state‐of‐the‐art of microfluidic technology, a robust platform for recapitulating complex cellular structure and function in synthetic models of biological cells.     

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales ^1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions^3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions^6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.     

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.     

A microfluidic array device for single cell capture and intracellular Ca2+ response analysis induced by dynamic biochemical stimulus

A microfluidic array was constructed for trapping single cell and loading identical dynamic biochemical stimulation for gain a better understanding of Ca²⁺ signaling at single cell resolution in the present study. This microfluidic array consists of multiple radially aligned flow channels with equal intersection angles, which was designed by a combination of stagnation point flow and physical barrier. Numerical simulation results and trajectory analysis have shown the effectiveness of this single cell trapping device. Fluorescent experiment results demonstrated the effects of flow rate and frequency of dynamic stimulus on the profiles of biochemical concentration which exposed on captured cells. In this microarray, the captured single cells in each trapping channels were able to receive identical extracellular dynamic biochemical stimuli which being transmitted from the entrance in the middle of the microfluidic array. Besides, after loading dynamic Adenosine Triphosphate (ATP) stimulation on captured cells by this device, consistent average intracellular Ca²⁺ dynamics phase and cellular heterogeneity were observed in captured single K562 cells. Furthermore, this device is able to be used for investigating cellular respond on single cell resolution to temporally varying environments by modulating the stimulation signal in terms of concentration, pattern, and duration of exposure.     

SuperSegger: robust image segmentation,analysis and lineage tracking of bacterial cells

Many quantitative cell biology questions require fast yet reliable automated image segmentation to identify and link cells from frame‐to‐frame, and characterize the cell morphology and fluorescence. We present SuperSegger, an automated MATLAB‐based image processing package well‐suited to quantitative analysis of high‐throughput live‐cell fluorescence microscopy of bacterial cells. SuperSegger incorporates machine‐learning algorithms to optimize cellular boundaries and automated error resolution to reliably link cells from frame‐to‐frame. Unlike existing packages, it can reliably segment microcolonies with many cells, facilitating the analysis of cell‐cycle dynamics in bacteria as well as cell‐contact mediated phenomena. This package has a range of built‐in capabilities for characterizing bacterial cells, including the identification of cell division events, mother, daughter and neighbouring cells, and computing statistics on cellular fluorescence, the location and intensity of fluorescent foci. SuperSegger provides a variety of postprocessing data visualization tools for single cell and population level analysis, such as histograms, kymographs, frame mosaics, movies and consensus images. Finally, we demonstrate the power of the package by analyzing lag phase growth with single cell resolution.     

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.     

Reproducible fashion of the HSP70B' promoter‐induced cytotoxic response on a live cell‐based biosensor by cell cycle synchronization

Live cell‐based sensors potentially provide functional information about the cytotoxic effect of reagents on various signaling cascades. Cells transfected with a reporter vector derived from a cytotoxic response promoter can be used as intelligent cytotoxicity sensors (i.e., sensor cells). We have combined sensor cells and a microfluidic cell culture system that can achieve several laminar flows, resulting in a reliable high‐throughput cytotoxicity detection system. These sensor cells can also be applied to single cell arrays. However, it is difficult to detect a cellular response in a single cell array, due to the heterogeneous response of sensor cells. The objective of this study was cell homogenization with cell cycle synchronization to enhance the response of cell‐based biosensors. Our previously established stable sensor cells were brought into cell cycle synchronization under serum‐starved conditions and we then investigated the cadmium chloride‐induced cytotoxic response at the single cell level. The GFP positive rate of synchronized cells was approximately twice as high as that of the control cells, suggesting that cell homogenization is an important step when using cell‐based biosensors with microdevices, such as a single cell array. Biotechnol. Bioeng. 2010;107: 561–565. © 2010 Wiley Periodicals, Inc.