In Situ Single‐Cell Western Blot on Adherent Cell Culture

Integrating 2D culture of adherent mammalian cells with single‐cell western blotting (in situ scWB) uses microfluidic design to eliminate the requirement for trypsin release of cells to suspension, prior to single‐cell isolation and protein analysis. To assay HeLa cells from an attached starting state, we culture adherent cells in fibronectin‐functionalized microwells formed in a thin layer of polyacrylamide gel. To integrate the culture, lysis, and assay workflow, we introduce a one‐step copolymerization process that creates protein‐decorated microwells. After single‐cell culture, we lyse each cell in the microwell and perform western blotting on each resultant lysate. We observe cell spreading after overnight microwell‐based culture. scWB reports increased phosphorylation of MAP kinases (ERK1/2, p38) under hypertonic conditions. We validate the in situ scWB with slab‐gel western blot, while revealing cell‐to‐cell heterogeneity in stress responses.     

Morphometric Cell Classification for Single‐Cell MALDI‐Mass Spectrometry Imaging

The large‐scale and label‐free molecular characterization of single cells in their natural tissue habitat remains a major challenge in molecular biology. We present a method that integrates morphometric image analysis to delineate and classify individual cells with their single‐cell‐specific molecular profiles. This approach provides a new means to study spatial biological processes such as cancer field effects and the relationship between morphometric and molecular features.     

Polyproline as a Minimal Antifreeze Protein Mimic That Enhances the Cryopreservation of Cell Monolayers

Tissue engineering, gene therapy, drug screening, and emerging regenerative medicine therapies are fundamentally reliant on high‐quality adherent cell culture, but current methods to cryopreserve cells in this format can give low cell yields and require large volumes of solvent “antifreezes”. Herein, we report polyproline as a minimum (bio)synthetic mimic of antifreeze proteins that is accessible by solution, solid‐phase, and recombinant methods. We demonstrate that polyproline has ice recrystallisation inhibition activity linked to its amphipathic helix and that it enhances the DMSO cryopreservation of adherent cell lines. Polyproline may be a versatile additive in the emerging field of macromolecular cryoprotectants.     

Dynamic single cell culture array

It is important to quantify the distribution of behavior amongst a population of individual cells to reach a more complete quantitative understanding of cellular processes. Improved high-throughput analysis of single cell behavior requires uniform conditions for individual cells with controllable cell-cell interactions, including diffusible and contact elements. Uniform cell arrays for static culture of adherent cells have previously been constructed using protein micropatterning techniques but lack the ability to control diffusible secretions. Here we present a microfluidic-based dynamic single cell culture array that allows both arrayed culture of individual adherent cells and dynamic control of fluid perfusion with uniform environments for individual cells. In our device no surface modification is required and cell loading is done in less than 30 seconds. The device consists of arrays of physical U-shaped hydrodynamic trapping structures with geometries that are biased to trap only single cells. HeLa cells were shown to adhere at a similar rate in the trapping array as on a control glass substrate. Additionally, rates of cell death and division were comparable to the control experiment. Approximately 100 individual isolated cells were observed growing and adhering in a field of view spanning approximately 1 mm(2) with greater than 85% of cells maintained within the primary trapping site after 24 hours. Also, greater than 90% of cells were adherent and only 5% had undergone apoptosis after 24 hours of perfusion culture within the trapping array. We anticipate uses in single cell analysis of drug toxicity with physiologically relevant perfused dosages as well as investigation of cell signaling pathways and systems biology.     

Current Advances in Highly Multiplexed Antibody‐Based Single‐Cell Proteomic Measurements

Single‐cell measurements have played a critical role in revealing the complex signaling dynamics and heterogeneity present in cells, but there is still much to learn. Measuring samples from bulk populations of cells often masks the information and dynamics present in subsets of cells. Common single‐cell protein studies rely on fluorescent microscopy and flow cytometry but are limited in multiplexing ability owing to spectral overlap. Recently, technology advancements in single‐cell proteomics have allowed highly multiplexed measurement of multiple parameters simultaneously by using barcoded microfluidic enzyme‐linked immunosorbent assays and mass cytometry techniques. In this review, we will describe recent work around multiparameter single‐cell protein measurements and critically analyze the techniques.     

Cell culture chip using low-shear mass transport

We have developed a flow cell that allows culturing adherent cells as well as suspended cells in a stable, homogeneous, and low-shear force environment. The device features continuous medium supply and waste exchange. In this paper, a simple and fast protocol for device design, fabrication, and assembly (sealing) based on a poly(dimethylsiloxane) (PMDS)/glass slide hybrid structure is described. The cell culture system performance was monitored, and the effective shear force inside the culture well was also determined. By manipulating the device dimensions and volumetric flow rate, shear stress was controlled during experiments. Cell adhesion, growth, proliferation, and death over long-term culture periods were observed by microscopy. The growth of both endothelial and suspension cells in this device exhibited comparable characteristics to those of traditional approaches. The low-shear culture device significantly reduced shear stress encountered in microfluidic systems, allowing both adherent and suspended cells to be grown in a simple device.     

Detection of LacZ‐Positive Cells in Living Tissue with Single‐Cell Resolution

The LacZ gene, which encodes Escherichia coli β‐galactosidase, is widely used as a marker for cells with targeted gene expression or disruption. However, it has been difficult to detect lacZ‐positive cells in living organisms or tissues at single‐cell resolution, limiting the utility of existing lacZ reporters. Herein we present a newly developed fluorogenic β‐galactosidase substrate suitable for labeling live cells in culture, as well as in living tissues. This precisely functionalized fluorescent probe exhibited dramatic activation of fluorescence upon reaction with the enzyme, remained inside cells by anchoring itself to intracellular proteins, and provided single‐cell resolution. Neurons labeled with this probe preserved spontaneous firing, which was enhanced by application of ligands of receptors expressed in the cells, suggesting that this probe would be applicable to investigate functions of targeted cells in living tissues and organisms.     

Probing Low‐Copy‐Number Proteins in a Single Living Cell

Single‐cell analysis techniques are essential for understanding the microheterogeneity and functions of cells. Low‐copy‐number proteins play important roles in cell functioning, but their measurement in single cells remains challenging. Herein, we report an approach, called plasmonic immunosandwich assay (PISA), for probing low‐copy‐number proteins in single cells. This approach combined in vivo immunoaffinity extraction and plasmon‐enhanced Raman scattering (PERS). Target proteins were specifically extracted from the cells by microprobes modified with monoclonal antibody or molecularly‐imprinted polymer (MIP), followed by labeling with Raman‐active nanotags. The PERS detection, with Raman intensity enhanced by 9 orders of magnitude, provided ultrasensitive detection at the single‐molecule level. Using this approach, we found that alkaline phosphatase and survivin were expressed in distinct levels in cancer and normal cells, and that extended culture passage resulted in reduced expression of survivin. We further developed acupuncture needle‐based PISA for probing low‐copy‐number proteins in living bodies.     

Microfluidic Device Using a Gold Pillar Array and Integrated Electrodes for On‐chip Endothelial Cell Immobilization,Direct RBC Contact,and Amperometric Detection of Nitric Oxide

We describe a microfluidic device that can be used to detect interactions between red blood cells (RBCs) and endothelial cells using a gold pillar array (created by electrodeposition) and an integrated detection electrode. Endothelial cells can release nitric oxide (NO) via stimulation by RBC‐derived ATP. These studies incorporate on‐chip endothelial cell immobilization, direct RBC contact, and detection of NO in a single microfluidic device. In order to study the RBC‐EC interactions, this work used a microfluidic device made of a PDMS chip with two adjacent channels and a polystyrene base with embedded electrodes for creating a membrane (via gold pillars) and detecting NO (at a glassy carbon electrode coated with platinum‐black and Nafion). RBCs were pharmacologically treated with treprostinil in the absence and presence of glybenclamide, and ATP release was determined as was the resultant NO release from endothelial cells. Treprostinil treatment of RBCs resulted in ATP release that stimulated endothelial cells to release on average 1.8±0.2 nM NO per endothelial cell (average±SEM, n=8). Pretreatment of RBCs with glybenclamide inhibited treprostinil‐induced ATP release and, therefore, less NO was produced by the endothelial cells (0.92±0.1 nM NO per endothelial cell, n=7). In the future, this device can be used to study interactions between many other cell types (both adherent and non‐adherent cell lines) and incorporate other detection schemes.     

Coating Strategies Using Layer‐by‐layer Deposition for Cell Encapsulation

The layer‐by‐layer (LbL) deposition technique is widely used to develop multilayered films based on the directed assembly of complementary materials. In the last decade, thin multilayers prepared by LbL deposition have been applied in biological fields, namely, for cellular encapsulation, due to their versatile processing and tunable properties. Their use was suggested as an alternative approach to overcome the drawbacks of bulk hydrogels, for endocrine cells transplantation or tissue engineering approaches, as effective cytoprotective agents, or as a way to control cell division. Nanostructured multilayered materials are currently used in the nanomodification of the surfaces of single cells and cell aggregates, and are also suitable as coatings for cell‐laden hydrogels or other biomaterials, which may later be transformed to highly permeable hollow capsules. In this Focus Review, we discuss the applications of LbL cell encapsulation in distinct fields, including cell therapy, regenerative medicine, and biotechnological applications. Insights regarding practical aspects required to employ LbL for cell encapsulation are also provided.     

微流控芯片上细胞培养与分析方法研究进展

微流控芯片以其强大的微流体和微小物质控制能力成为研究单细胞、细胞群落乃至生物组织的重要手段。在本篇综述中,我们将以微流控芯片上细胞体外培养模型的建立为主,对近几年来重要的研究工作加以评述,全面地介绍微流控技术在细胞生命科学研究中应用的优势和未来发展方向,具体包括微流控芯片的细胞操控能力、细胞培养微环境的构建以及芯片联用检测手段,希望为从事这一领域研究工作的读者提供一些新的思路。     

Chemical and Topographical Single‐Cell Imaging by Near‐Field Desorption Mass Spectrometry

Simultaneously acquiring chemical and topographical information within a single cell at nanoscale resolutions is vital to cellular biology, yet it remains a great challenge due to limited lateral resolutions and detection sensitivities. Herein, the development of near‐field desorption mass spectrometry for correlated chemical and topographical imaging is reported, thereby bridging the gap between laser‐based mass spectrometry (MS) methods and multimodal single‐cell imaging. Using this integrated platform, an imaging resolution of 250 nm and 3D topographically reconstructed chemical single‐cell imaging were achieved. This technique offers more in‐depth cellular information than micrometer‐range laser‐based MS imaging methods. Considering the simplicity and compact size of the near‐field device, this technique can be introduced to MALDI‐MS, expanding the multimodal abilities of MS at nanoscale resolutions.     

Highly Multiplexed Single‐Cell In Situ Protein Analysis with Cleavable Fluorescent Antibodies

Limitations on the number of proteins that can be quantified in single cells in situ impede advances in our deep understanding of normal cell physiology and disease pathogenesis. Herein, we present a highly multiplexed single‐cell in situ protein analysis approach that is based on chemically cleavable fluorescent antibodies. In this method, antibodies tethered to fluorophores through a novel azide‐based cleavable linker are utilized to detect their protein targets. After fluorescence imaging and data storage, the fluorophores coupled to the antibodies are efficiently cleaved without loss of protein target antigenicity. Upon continuous cycles of target recognition, fluorescence imaging, and fluorophore cleavage, this approach has the potential to quantify over 100 different proteins in individual cells at optical resolution. This single‐cell in situ protein profiling technology will have wide applications in signaling network analysis, molecular diagnosis, and cellular targeted therapies.     

High‐Throughput Isolation of Cell Protrusions with Single‐Cell Precision for Profiling Subcellular Gene Expression

Invading cancer cells extend cell protrusions, which guide cancer‐cell migration and invasion, eventually leading to metastasis. The formation and activity of cell protrusions involve the localization of molecules and organelles at the cell front; however, it is challenging to precisely isolate these subcellular structures at the single‐cell level for molecular analysis. Here, we describe a newly developed microfluidic platform capable of high‐throughput isolation of cell protrusions at single‐cell precision for profiling subcellular gene expression. Using this microfluidic platform, we demonstrate the efficient generation of uniform cell‐protrusion arrays (more than 5000 cells with protrusions) for a series of cell types. We show precise isolation of cell protrusions with high purity at single‐cell precision for subsequent RNA‐Seq analysis, which was further validated by RT‐qPCR and RNA FISH. Our highly controlled protrusion isolation method opens a new avenue for the study of subcellular functional mechanisms and signaling pathways in metastasis.     

Plasmonic Surfaces for Cell Growth and Retrieval Triggered by Near‐Infrared Light

Methods for efficient detachment of cells avoiding damage are required in tissue engineering and regenerative medicine. We introduce a bottom–up approach to build plasmonic substrates using micellar block copolymer nanolithography to generate a 2D array of Au seeds, followed by chemical growth leading to anisotropic nanoparticles. The resulting plasmonic substrates show a broad plasmon band covering a wide part of the visible and near‐infrared (NIR) spectral ranges. Both human and murine cells were successfully grown on the substrates. A simple functionalization step of the plasmonic substrates with the cyclic arginylglycylaspartic acid (c‐RGD) peptide allowed us to tune the morphology of integrin‐rich human umbilical vein endothelial cells (HUVEC). Subsequent irradiation with a NIR laser led to highly efficient detachment of the cells with cell viability confirmed using the MTT assay. We thus propose the use of such plasmonic substrates for cell growth and controlled detachment using remote near‐IR irradiation, as a general method for cell culture in biomedical applications.     

Multivalent Chelators for In Vivo Protein Labeling

With the advent of single‐molecule methods, chemoselective and site‐specific labeling of proteins evolved to become a central aspect in chemical biology as well as cell biology. Protein labeling demands high specificity, rapid as well as efficient conjugation, while maintaining low concentration and biocompatibility under physiological conditions. Generic methods that do not interfere with the function, dynamics, subcellular localization of proteins, and crosstalk with other factors are crucial to probe and image proteins in vitro and in living cells. Alternatives to enzyme‐based tags or autofluorescent proteins are short peptide‐based recognition tags. These tags provide high specificity, enhanced binding rates, bioorthogonality, and versatility. Here, we report on recent applications of multivalent chelator heads, recognizing oligohistidine‐tagged proteins. The striking features of this system has facilitated the analysis of protein complexes by single‐molecule approaches.     

Selective Adhesion and Directional Migration of Endothelial Cells Guided by Cys‐Ala‐Gly Peptide Density Gradient on Antifouling Polymer Brushes

Selective adhesion and directional migration of endothelial cells (ECs) on biomaterials is critical to realize the rapid endothelialization. In this study, a Cys‐Ala‐Gly (CAG) peptide density gradient is generated on homogeneous cell‐resisting poly(2‐hydroxyethyl methacrylate‐co‐glycidyl methacrylate) brushes by immersing the brushes in a complementary gradient solution of CAG and competitive mercapto‐terminated methoxyl poly(ethylene glycol). The adhesion and spreading of smooth muscle cells (SMCs) is impaired effectively on the gradient surface. About six folds of adherent ECs over SMCs are achieved at the position (10 mm) of highest CAG density on the gradient surface in a co‐culture condition. Due to the gradient cues, ECs migrate fastest with the best directionality of 86.7% at the middle of the gradient, leading to the maximum net displacement as well.     

A Versatile Dynamic Mussel‐Inspired Biointerface: From Specific Cell Behavior Modulation to Selective Cell Isolation

Reported here is a novel dynamic biointerface based on reversible catechol‐boronate chemistry. Biomimetically designed peptides with a catechol‐containing sequence and a cell‐binding sequence at each end were initially obtained. The mussel‐inspired peptides were then reversibly bound to a phenylboronic acid (PBA) containing polymer‐grafted substrate through sugar‐responsive catechol‐boronate interactions. The resultant biointerface is thus capable of dynamic presentation of the bioactivity (i.e. the cell‐binding sequence) by virtue of changing sugar concentrations in the system (similar to human glycemic volatility). In addition, the sugar‐responsive biointerface enables not only dynamic modulation of stem cell adhesion behaviors but also selective isolation of tumor cells. Considering the highly biomimetic nature and biological stimuli‐responsiveness, this mussel‐inspired dynamic biointerface holds great promise in both fundamental cell biology research and advanced medical applications.     

High‐Spatial Resolution Mass Spectrometry Imaging: Toward Single Cell Metabolomics in Plant Tissues

Mass spectrometry imaging (MSI) is a powerful tool that has advanced our understanding of complex biological processes by enabling unprecedented details of metabolic biology to be uncovered. Through the use of high‐spatial resolution MSI, metabolite localizations can be obtained with high precision. Here we describe our recent progress to enhance the spatial resolution of matrix‐assisted laser desorption/ionization (MALDI) MSI from ∼50 μm with the commercial configuration to ∼5 μm. Additionally, we describe our efforts to develop a ‘multiplex MSI’ data acquisition method to allow more chemical information to be obtained on a single tissue in a single instrument run, and the development of new matrices to improve the ionization efficiency for a variety of small molecule metabolites. In combination, these contributions, along with the efforts of others, will bring MSI experiments closer to achieving metabolomic scale.     

Intercellular Connections Related to Cell–Cell Crosstalk Specifically Recognized by an Aptamer

Intercellular connections are an important pathway for cell–cell crosstalk. However, their formation mechanism and functions are far from being understood. The lack of molecular probes hampers the research in this area. Herein, we report a kind of intercellular connection that is specifically recognized by aptamer M17A2 generated by cell‐SELEX against MCF‐7R cells. These connections have different morphologies, but have the same skeleton composed of F‐actin. The long filamentous connections were identified to be tunneling nanotubes (TNTs), a recently discovered cell–cell communication route. These connections could be built not only between MCF‐7R cells, but also from MCF‐7R to other cells after co‐culture. Proteins could be transported between cells through these connections, suggesting their cell communication function. Aptamer M17A2 shows the potential to act as a new probe for investigating this kind of intercellular connection, as well as for studying cell–cell communication.