A method for isolating contractile smooth muscle cells from cryopreserved tissue.

Multiple techniques have been developed to isolate contractile smooth muscle cells (SMCs) from tissues with varying degrees of success. However, most of these approaches rely on obtaining fresh tissue, which poses logistical challenges. In the present study, we introduce a novel protocol for isolating contractile SMCs from cryopreserved smooth muscle (SM) tissue, thereby enhancing experimental efficiency. This protocol yields abundant viable, spindle-shaped, contractile SMCs that closely resemble those obtained from fresh samples. By analyzing the expression of contractile proteins, we demonstrate that both the isolated SMCs from cryopreserved tissue represent more accurately fresh SM tissue compared with cultured SMCs. Moreover, we demonstrate the importance of a brief incubation step of the tissue in culture medium before cell dissociation to achieve contractile SMCs. Finally, we provide a concise overview of our protocol optimization efforts, along with a summary of previously published methods, which could be valuable for the development of similar protocols for other species.NEW & NOTEWORTHY We report a successful protocol development for isolating contractile smooth muscle cells (SMCs) from cryopreserved tissue reducing the reliance on fresh tissues and providing a readily available source of contractile SMCs. Our findings suggest that SMCs isolated using our protocol maintain their phenotype better compared with cultured SMCs. This preservation of the cellular characteristics, including the expression of key contractile proteins, makes these cells more representative of fresh SM tissue.

A guide to epigenetics in leukaemia stem cells.

Leukaemia stem cells (LSCs) are the critical seed for the growth of haematological malignancies, driving the clonal expansion that enables disease initiation, relapse and often resistance. Specifically, they display inherent phenotypic and epigenetic plasticity resulting in complex heterogenic diseases. In this review, we discuss the key principles of deregulation of epigenetic processes that shape this disease evolution. We consider measures to define and quantify clonal heterogeneity, combining information from recent studies assessing mutational, transcriptional and epigenetic landscapes at single cell resolution in myeloid neoplasms (MN). We highlight the importance of integrating epigenetic and genetic information to better understand inter- and intra-patient heterogeneity and discuss how this understanding further informs evolution and progression trajectories and subsequent clinical response in MN. Under this topic, we also discuss efforts to identify mechanisms of resistance, by longitudinal analyses of patient samples. Finally, we highlight how we might target these aberrant epigenetic processes for better therapeutic outcomes and to potentially eradicate LSCs.

Single-Cell Profiling Reveals Functional Heterogeneity and Serial Killing in Human Peripheral and Ex Vivo-Generated CD34+ Progenitor-Derived Natural Killer Cells.

Increasing evidence suggests that natural killer (NK) cells are composed of distinct functional subsets. This multifunctional role has made them an attractive choice for anticancer immunotherapy. A functional NK cell repertoire is generated through cellular education, resulting in a heterogeneous NK cell population with distinct capabilities responding to different stimuli. The application of a high-throughput droplet-based microfluidic platform allows monitoring of NK cell-target cell interactions at the single-cell level and in real-time. A variable response of single NK cells toward different target cells is observed, and a distinct population of NK cells (serial killers) capable of inducing multiple target lysis is identified. By assessing the cytotoxic dynamics, it is shown that single umbilical cord blood-derived CD34+ hematopoietic progenitor (HPC)-NK cells display superior antitumor cytotoxicity. With an integrated analysis of cytotoxicity and cytokine secretion, it is shown that target cell interactions augment cytotoxic as well as secretory behavior of NK cells. By providing an integrated assessment of NK cell functions by microfluidics, this study paves the way to further functionally characterize NK cells ultimately aimed to improve cancer immunotherapy.

The transcription factor Atf1 lowers the transition barrier for nucleosome-mediated establishment of heterochromatin.

Transcription factors can exert opposite effects depending on the chromosomal context. The fission yeast transcription factor Atf1 both activates numerous genes in response to stresses and mediates heterochromatic gene silencing in the mating-type region. Investigating this context dependency, we report here that the establishment of silent heterochromatin in the mating-type region occurs at a reduced rate in the absence of Atf1 binding. Quantitative modeling accounts for the observed establishment profiles by a combinatorial recruitment of histone-modifying enzymes: locally by Atf1 at two binding sites and over the whole region by dynamically appearing heterochromatic nucleosomes, a source of which is the RNAi-dependent cenH element. In the absence of Atf1 binding, the synergy is lost, resulting in a slow rate of heterochromatin formation. The system shows how DNA-binding proteins can influence local nucleosome states and thereby potentiate long-range positive feedback on histone-modification reactions to enable heterochromatin formation over large regions in a context-dependent manner.

Optimization and Fabrication of Multi-Level Microchannels for Long-Term Imaging of Bacterial Growth and Expansion.

Bacteria are unicellular organisms whose length is usually around a few micrometers. Advances in microfabrication techniques have enabled the design and implementation of microdevices to confine and observe bacterial colony growth. Microstructures hosting the bacteria and microchannels for nutrient perfusion usually require separate microfabrication procedures due to different feature size requirements. This fact increases the complexity of device integration and assembly process. Furthermore, long-term imaging of bacterial dynamics over tens of hours requires stability in the microscope focusing mechanism to ensure less than one-micron drift in the focal axis. In this work, we design and fabricate an integrated multi-level, hydrodynamically-optimized microfluidic chip to study long-term Escherichia coli population dynamics in confined microchannels. Reliable long-term microscopy imaging and analysis has been limited by focus drifting and ghost effect, probably caused by the shear viscosity changes of aging microscopy immersion oil. By selecting a microscopy immersion oil with the most stable viscosity, we demonstrate successful captures of focally stable time-lapse bacterial images for ≥72 h. Our fabrication and imaging methodology should be applicable to other single-cell studies requiring long-term imaging.

Electrochemiluminescence Loss in Photobleaching.

The effects of photobleaching on electrochemiluminescence (ECL) was investigated for the first time. The plasma membrane of Chinese Hamster Ovary (CHO) cells was labeled with a [Ru(bpy)3 ]2+ derivative. Selected regions of the fixed cells were photobleached using the confocal mode with sequential stepwise illumination or cumulatively and they were imaged by both ECL and photoluminescence (PL). ECL was generated with a model sacrificial coreactant, tri-n-propylamine. ECL microscopy of the photobleached regions shows lower ECL emission. We demonstrate a linear correlation between the ECL decrease and the PL loss due to the photobleaching of the labels immobilized on the CHO membranes. The presented strategy provides valuable information on the fundamentals of the ECL excited state and opens new opportunities for exploring cellular membranes by combining ECL microscopy with photobleaching techniques such as fluorescence recovery after photobleaching (FRAP) or fluorescence loss in photobleaching (FLIP) methods.

Bio-Coreactant-Enhanced Electrochemiluminescence Microscopy of Intracellular Structure and Transport.

A bio-coreactant-enhanced electrochemiluminescence (ECL) microscopy realizes the ECL imaging of intracellular structure and dynamic transport. This microscopy uses Ru(bpy)32+ as the electrochemical molecular antenna connecting extracellular and intracellular environments, and uses intracellular biomolecules as the coreactants of ECL reactions via a "catalytic route". Accordingly, intracellular structures are identified without using multiple labels, and autophagy involving DNA oxidative damage is detected using nuclear ECL signals. A time-resolved image sequence discloses the universal edge effect of cellular electroporation due to the influence of the geometric properties of cell membranes on the induced transmembrane voltage. The dynamic transport of Ru(bpy)33+ in the different cellular compartments unveils the heterogeneous intracellular diffusivity correlating with the actin cytoskeleton. In addition to single-cell studies, the bio-coreactant-enhanced ECL microscopy is used to image a slice of a mouse liver and a colony of Shewanella oneidensis MR-1.

Scaffold-free biofabrication of adipocyte structures with magnetic levitation.

Tissue engineering research aims to repair the form and/or function of impaired tissues. Tissue engineering studies mostly rely on scaffold-based techniques. However, these techniques have certain challenges, such as the selection of proper scaffold material, including mechanical properties, sterilization, and fabrication processes. As an alternative, we propose a novel scaffold-free adipose tissue biofabrication technique based on magnetic levitation. In this study, a label-free magnetic levitation technique was used to form three-dimensional (3D) scaffold-free adipocyte structures with various fabrication strategies in a microcapillary-based setup. Adipogenic-differentiated 7F2 cells and growth D1 ORL UVA stem cells were used as model cells. The morphological properties of the 3D structures of single and cocultured cells were analyzed. The developed procedure leads to the formation of different patterns of single and cocultured adipocytes without a scaffold. Our results indicated that adipocytes formed loose structures while growth cells were tightly packed during 3D culture in the magnetic levitation platform. This system has potential for ex vivo modeling of adipose tissue for drug testing and transplantation applications for cell therapy in soft tissue damage. Also, it will be possible to extend this technique to other cell and tissue types.

Integrity of a heterochromatic domain ensured by its boundary elements.

In fission yeast, the inverted repeats IR-L and IR-R function as boundary elements at the edges of a 20-kb silent heterochromatic domain where nucleosomes are methylated at histone H3K9. Each repeat contains a series of B-box motifs physically associated with the architectural TFIIIC complex and with other factors including the replication regulator Sap1 and the Rix1 complex (RIXC). We demonstrate here the activity of these repeats in heterochromatin formation and maintenance. Deletion of the entire IR-R repeat or, to a lesser degree, deletion of just the B boxes impaired the de novo establishment of the heterochromatic domain. Nucleation proceeded normally at the RNA interference (RNAi)-dependent element cenH but subsequent propagation to the rest of the region occurred at reduced rates in the mutants. Once established, heterochromatin was unstable in the mutants. These defects resulted in bistable populations of cells occupying alternate "on" and "off" epigenetic states. Deleting IR-L in combination with IR-R synergistically tipped the balance toward the derepressed state, revealing a concerted action of the two boundaries at a distance. The nuclear rim protein Amo1 has been proposed to tether the mating-type region and its boundaries to the nuclear envelope, where Amo1 mutants displayed milder phenotypes than boundary mutants. Thus, the boundaries might facilitate heterochromatin propagation and maintenance in ways other than just through Amo1, perhaps by constraining a looped domain through pairing.

Electrochemiluminescence Single-Cell Analysis: Intensity- and Imaging-Based Methods.

Cellular heterogeneity is always present in any cell population. It is the fundamental to understand how single cells and cell ensembles behave under perturbation, which is essential for both basic research and clinical application. Various approaches have been developed to study cell-to-cell difference at the single-cell level, among which electrochemiluminescence (ECL) single-cell analysis has emerged in recent years. This Minireview focuses on the advances in ECL single cell analysis. After a brief introduction to single cell analysis and ECL, this overview mainly covers the ECL mechanisms and systems involved in ECL cytosensors, as well as the applications in ECL single cell analysis, which are classified into two main categories, namely intensity- and imaging-based methods. Finally, the outlooks and challenges in this field are discussed.

Imaging Cell-Matrix Adhesions and Collective Migration of Living Cells by Electrochemiluminescence Microscopy.

Cell-matrix adhesions play essential roles in a variety of biological processes. Herein, we report a label-free method to map cell-matrix adhesions of single living cells on an electrode surface by electrochemiluminescence (ECL). An indium tin oxide electrode modified with a silica nanochannel membrane was used as the substrate electrode, at which the ECL generation from freely diffusing luminophores provided a distinct visual contrast between adhesion sites and noncontacted domains, thus selectively revealing the former in a label-free manner. With this methodology, we studied the spatial distribution, as well as dynamic variation, of cell-matrix adhesions and the adhesion strength at the subcellular level. Cell-matrix adhesions of an advancing cell sheet were finally imaged to study the movement of cells in collective migration. A statistical analysis suggests that cells on the far side of leading edge also have the propensity to migrate and do not act as just passive followers.

Microfluidic technology for investigation of protein function in single adherent cells.

Instrumental techniques and associated methods for single cell analysis, designed to investigate and measure a broad range of cellular parameters in search of unique features, address key limitations of conventional cell-based assays with their ensemble average response. While many different single cell techniques exist for suspension cultures, which can process and characterize large numbers of individual cells in rapid succession, the access to surface-immobilized cells in typical 2D and 3D culture environments remains challenging. Open space microfluidics has created new possibilities in this area, allowing for exclusive access to single cells in adherent cultures, even at high confluency. In this chapter, we briefly review new microtechnologies for the investigation of protein function in single adherent cells, and present an overview over related recent applications of the multifunctional pipette (Biopen), a microfluidic multi-solution dispensing system that uses hydrodynamic confinement in open volume environments in order to establish a superfusion zone over selected single cells in adherent cultures.

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.

Transient Replication in Specialized Cells Favors Transfer of an Integrative and Conjugative Element.

Integrative and conjugative elements (ICEs) are widespread mobile DNA within bacterial genomes, whose lifestyle is relatively poorly understood. ICEs transmit vertically through donor cell chromosome replication, but in order to transfer, they have to excise from the chromosome. The excision step makes ICEs prone to loss, in case the donor cell divides and the ICE is not replicated. By adapting the system of LacI-cyan fluorescent protein (CFP) binding to lacO operator arrays, we analyze here the process of excision and transfer of the ICE for 3-chlorobenzoate degradation (ICEclc) in individual cells of the bacterium Pseudomonas putida We provide evidence that ICEclc excises exclusively in a subset of specialized transfer-competent cells. ICEclc copy numbers in transfer-competent cells were higher than in regular nontransferring cells but were reduced in mutants lacking the ICE oriT1 origin of transfer, the ICE DNA relaxase, or the excision recombination sites. Consistently, transfer-competent cells showed a higher proportion without any observable LacI-CFP foci, suggesting ICEclc loss, but this proportion was independent of the ICE relaxase or the ICE origins of transfer. Our results thus indicated that the excised ICE becomes transiently replicated in transfer-competent cells, with up to six observable copies from LacI-CFP fluorescent focus measurements. Most of the observed ICEclc transfer to ICE-free P. putida recipients occurred from donors displaying 3 to 4 ICE copies, which constitute a minority among all transfer-competent cells. This finding suggests, therefore, that replication of the excised ICEclc in donors is beneficial for transfer fitness to recipient cells.IMPORTANCE Bacterial evolution is driven to a large extent by horizontal gene transfer (HGT)-the processes that distribute genetic material between species rather than by vertical descent. The different elements and processes mediating HGT have been characterized in great molecular detail. In contrast, very little is known on adaptive features selecting HGT evolvability and fitness optimization. By studying the molecular behavior of an integrated mobile DNA of the class of integrative and conjugative elements in individual Pseudomonas putida donor bacteria, we report here how transient replication of the element after its excision from the chromosome is favorable for its transfer success. Since successful transfer into a new recipient is a measure of the element's fitness, transient replication may have been selected as an adaptive benefit for more-optimal transfer.

The ABC Guide to Fluorescent Toolsets for the Development of Future Biomaterials.

In recent decades, diversified approaches using nanoparticles or nano-structured scaffolds have been applied to drug delivery and tissue engineering. Thanks to recent interdisciplinary studies, the materials developed have been intensively evaluated at animal level. Despite these efforts, less attention has been paid to what is really going on at the subcellular level during the interaction between a nanomaterial and a cell. As the proposed concept becomes more complex, the need for investigation of the dynamics of these materials at the cellular level becomes more prominent. For a deeper understanding of cellular events, fluorescent imaging techniques have been a powerful means whereby spatiotemporal information related to cellular events can be visualized as detectable fluorescent signals. To date, several excellent review papers have summarized the use of fluorescent imaging toolsets in cellular biology. However, applying these toolsets becomes a laborious process for those who are not familiar with imaging studies to engage with owing to the skills gap between them and cell biologists. This review aims to highlight the valuable essentials of fluorescent imaging as a tool for the development of effective biomaterials by introducing some cases including photothermal and photodynamic therapies. This distilled information will be a convenient short-cut for those who are keen to fabricate next generation biomaterials.

Supervised classification of etoposide-treated in vitro adherent cells based on noninvasive imaging morphology.

Single-cell studies using noninvasive imaging is a challenging, yet appealing way to study cellular characteristics over extended periods of time, for instance to follow cell interactions and the behavior of different cell types within the same sample. In some cases, e.g., transplantation culturing, real-time cellular monitoring, stem cell studies, in vivo studies, and embryo growth studies, it is also crucial to keep the sample intact and invasive imaging using fluorophores or dyes is not an option. Computerized methods are needed to improve throughput of image-based analysis and for use with noninvasive microscopy such methods are poorly developed. By combining a set of well-documented image analysis and classification tools with noninvasive microscopy, we demonstrate the ability for long-term image-based analysis of morphological changes in single cells as induced by a toxin, and show how these changes can be used to indicate changes in biological function. In this study, adherent cell cultures of DU-145 treated with low-concentration (LC) etoposide were imaged during 3 days. Single cells were identified by image segmentation and subsequently classified on image features, extracted for each cell. In parallel with image analysis, an MTS assay was performed to allow comparison between metabolic activity and morphological changes after long-term low-level drug response. Results show a decrease in proliferation rate for LC etoposide, accompanied by changes in cell morphology, primarily leading to an increase in cell area and textural changes. It is shown that changes detected by image analysis are already visible on day 1 for [Formula: see text] etoposide, whereas effects on MTS and viability are detected only on day 3 for [Formula: see text] etoposide concentration, leading to the conclusion that the morphological changes observed occur before and at lower concentrations than a reduction in cell metabolic activity or viability. Three classifiers are compared and we report a best case sensitivity of 88% and specificity of 94% for classification of cells as treated/untreated.

Ratiometric analysis of Acridine Orange staining in the study of acidic organelles and autophagy.

Acridine Orange is a cell-permeable green fluorophore that can be protonated and trapped in acidic vesicular organelles (AVOs). Its metachromatic shift to red fluorescence is concentration-dependent and, therefore, Acridine Orange fluoresces red in AVOs, such as autolysosomes. This makes Acridine Orange staining a quick, accessible and reliable method to assess the volume of AVOs, which increases upon autophagy induction. Here, we describe a ratiometric analysis of autophagy using Acridine Orange, considering the red-to-green fluorescence intensity ratio (R/GFIR) to quantify flow cytometry and fluorescence microscopy data of Acridine-Orange-stained cells. This method measured with accuracy the increase in autophagy induced by starvation or rapamycin, and the reduction in autophagy produced by bafilomycin A1 or the knockdown of Beclin1 or ATG7. Results obtained with Acridine Orange, considering R/GFIR, correlated with the conversion of the unlipidated form of LC3 (LC3-I) into the lipidated form (LC3-II), SQSTM1 degradation and GFP-LC3 puncta formation, thus validating this assay to be used as an initial and quantitative method for evaluating the late step of autophagy in individual cells, complementing other methods.

Single-Cell Mass Spectrometry for Discovery Proteomics: Quantifying Translational Cell Heterogeneity in the 16-Cell Frog (Xenopus) Embryo.

We advance mass spectrometry from a cell population-averaging tool to one capable of quantifying the expression of diverse proteins in single embryonic cells. Our instrument combines capillary electrophoresis (CE), electrospray ionization, and a tribrid ultrahigh-resolution mass spectrometer (HRMS) to enable untargeted (discovery) proteomics with ca. 25 amol lower limit of detection. CE-µESI-HRMS enabled the identification of 500-800 nonredundant protein groups by measuring 20 ng, or <0.2% of the total protein content in single blastomeres that were isolated from the 16-cell frog (Xenopus laevis) embryo, amounting to a total of 1709 protein groups identified between n=3 biological replicates. By quantifying ≈150 nonredundant protein groups between all blastomeres and replicate measurements, we found significant translational cell heterogeneity along multiple axes of the embryo at this very early stage of development when the transcriptional program of the embryo has yet to begin.

Three-dimensional mid-infrared tomographic imaging of endogenous and exogenous molecules in a single intact cell with subcellular resolution.

Microscopy in the mid-infrared spectral range provides detailed chemical information on a sample at moderate spatial resolution and is being used increasingly in the characterization of biological entities as challenging as single cells. However, a conventional cellular 2D imaging measurement is limited in its ability to associate specific compositional information to subcellular structures because of the interference from the complex topography of the sample. Herein we provide a method and protocols that overcome this challenge in which tilt-series infrared tomography is used with a standard benchtop infrared microscope. This approach gives access to the quantitative 3D distribution of molecular components based on the intrinsic contrast provided by the sample. We demonstrate the method by quantifying the distribution of an exogenous metal carbonyl complex throughout the cell and by reporting changes in its coordination sphere in different locations in the cell.