Platforms for Single-Cell Collection and Analysis

In adipocytes, insulin triggers the redistribution of Glut4 from intracellular compartments to the plasma membrane. Two models have been proposed to explain the effect of insulin on Glut4 localization. In the first, termed dynamic exchange, Glut4 continually cycles between the plasma membrane and intracellular compartments in basal cells, and the major effect of insulin is through changes in the exocytic and endocytic rate constants, k(ex) and k(en). In the second model, termed static retention, Glut4 is packaged in specialized storage vesicles (GSVs) in basal cells and does not traffic through the plasma membrane or endosomes. Insulin triggers GSV exocytosis, increasing the amount of Glut4 in the actively cycling pool. Using a flow cytometry-based assay, we found that Glut4 is regulated by both static and dynamic retention mechanisms. In basal cells, 75-80% of the Glut4 is packaged in noncycling GSVs. Insulin increased the amount of Glut4 in the actively cycling pool 4-5-fold. Insulin also increased k(ex) in the cycling pool 3-fold. After insulin withdrawal, Glut4 is rapidly cleared from the plasma membrane (t((1/2)) of 20 min) by rapid adjustments in k(ex) and k(en) and recycled into static compartments. Complete recovery of the static pool required more than 3 h, however. We conclude that in fully differentiated confluent adipocytes, both the dynamic and static retention mechanisms are important for the regulation of plasma membrane Glut4 content. However, cell culture conditions affect Glut4 trafficking. For example, replating after differentiation inhibited the static retention of Glut4, which may explain differences in previous reports.

Study of kaempferol in the treatment of rheumatoid arthritis through modulation of the NLRP3/CASP1/GSDMD axis and T-cell activation: Based on network pharmacology, single-cell analysis, and experimental validation

<p>Droplet microfluidics is the science of manipulation of discrete volumes of fluid in the nanoliter to femtolitre range. One of the important applications of droplet microfluidics is cargoencapsulation. The droplets can be loaded with live cells or therapeutics, with applications in single-cell analysis or drug delivery, respectively. </p> <p>The conventional system in droplet microfluidics is water-oil, where droplets of water that encapsulate the cargo are generated in an immiscible phase of oil. Because of the presence of the non-biocompatible oil phase, this traditional two-phase system poses complications for biomedical applications. </p> <p>Over the past decade, aqueous two-phase systems (ATPS) have emerged as an alternative for the conventional water-oil systems. ATPSs are composed of immiscible aqueous solutions of two incompatible polymers or a polymer and a salt. Owing to the aqueous nature of both phases, these systems are biocompatible. </p> <p>In this thesis, I explain the development of a magnetophoretic all-aqueous droplet microfluidic platform by the integration of a commonly used polymeric ATPS and aqueous based ferrofluids—a colloidal suspension of iron oxide nanoparticles. I have applied this platform to two major applications of droplet microfluidics: single-cell analysis and drug delivery. </p> <p>In the first part of my thesis, I show that ATPS droplets can be functionalized with ferrofluid for magnetic droplet manipulation. I show control over droplet manipulation by changing various parameters in the system such as the magnetic field and ferrofluid concentration. Additionally, I modify an existing mathematical model which was previously developed for modeling the deflection of magnetic beads in microfluidics and apply it to my droplet microfluidic system and show a good agreement between the model and the experimental data. </p> <p>Next, I show that by coupling cell-triggered Rayleigh-Plateau instability and diamagnetism in an ATPS droplet microfluidic platform, a pure population of cell-encapsulating droplets can be generated. Such a pure sample of single-cell encapsulating droplets is an essential component of droplet-based single-cell analysis. I generate single-cell encapsulating droplets by passive methods and separate cell-encapsulating droplets from the empty waste droplets by size-based diamagnetic droplet sorting. I show that by changing the magnetic field, the separation efficiency of empty and cell-encapsulating droplets increases, and cells used in such a microfluidic system show a high level of viability. </p> <p>In the last part of my thesis, I use the magnetic droplet microfluidic platform, reported in the first part of the thesis, for the fabrication of magnetic polyelectrolyte microcapsules. Polyelectrolytes are polymers with an electrolyte group. Once dissolved in deionized water, they become charged. Using two oppositely charged polyelectrolytes in the two phases of an ATPS droplet microfluidic system, and by careful tuning of the concentrations, I fabricate magnetic polyelectrolyte microcapsules. Such microcapsules have applications in targeted drug delivery. Therefore, I characterize the delivery profile of these microcapsules using pseudo-drugs with different molecular weights and show triggered release of their cargo by exposing the microcapsules to different stimuli such as osmotic pressure and change in pH. </p> <p>This magnetophoretic all-aqueous droplet microfluidic platform will find applications in single-cell analysis and fabrication of functionalized drug carriers for targeted drug delivery.</p>

Cytometry is the characterization and measurement of cells and cellular constituents for biological, diagnostic and therapeutic purposes, embracing the fields of cell and molecular biology, biochemistry, biophysics, cell physiology, pathology, immunology, genetics, biotechnology, plant biology and microbiology. Cytometry is based on quantitative measurements of the molecular and phenotypic properties of cells using flow and image cytometry, microarrays, and proteomics. Cytomics (molecular cell systems research) aims at the understanding of the molecular architecture and functionality of cell systems (cytomes) by single-cell analysis in combination with exhaustive bioinformatic knowledge extraction. The cytomics concept has been significantly advanced by a multitude of current developments like confocal and laser scanning microscopy, multiphoton fluorescence excitation, spectral imaging, fluorescence resonance energy transfer (FRET), fast imaging in flow, optical stretching in flow, and miniaturized flow and image cytometry within laboratories on a chip or laser microdissection, as well as the use of bead arrays [1, 2]. Data sieving or data mining of the vast amounts of collected multiparameter data for exhaustive multilevel bioinformatic knowledge extraction avoids the inadvertent loss of information from unknown molecular relations being inaccessible to an a priori hypothesis. These approaches will become powerful tools for such important fields as individualized medicine, drug discovery and drug development [3–6]. This special issue is focused on state-of-the-art research in advanced cytometry and application areas with particular emphasis on novel biophotonic methods, disease diagnosis, and monitoring of disease treatment at single-cell level in stationary and flow conditions. It seeks to advance scholarly research that spans from fundamental interactions between light, cells, vascular tissue, and labeling particles, to strategies and opportunities for preclinical and clinical research. Recent advances in slide-based cytometry for in vitro application are summarized by Gerstner et al. [7]. They show that single cell based quantitation using microscope based cytometry instruments is making its way from basic research into clinical use. Calibration and quality control are essential in every cytometric analysis. Basics of standardization and calibration in cytometry are comprehensively summarized by Mittag and colleague [8]. Leif [9] demonstrates how data formats produced by cytometric measurements can be integrated into clinical diagnostic system standards for improved data management in the clinical environment. Flow cytometry (FCM) is based both on cell detection as whole and the use of high-speed imaging of individual cells and nanoparticles. Tychinski and co-workers [10] analyze the metabolic component of cellular refractivity and its importance for optical cytometry. The refractivity decrease in response to energy depletion was demonstrated for mitochondria, chloroplasts, spores, and cancer cells using the coherent phase microscope “Airyscan”. Estimations indicated high values for the equivalent refractive index of structured water in cells. To reduce the interference from tissue light scattering background, gold nanoparticles with strong plasmon scattering resonance properties can be applied as in vivo FCM contrast agents. This technique requires an understanding of light interaction features of cells with and without nanoparticles. Tanev and coworkers [11] compare light scattering of these cells in controlled refractive index matching conditions. They analyze the optical schematics including phase contrast microscopy as a prospective modality for in vivo FCM. Jacobs et al. [12] consider some important features of the diffraction imaging of spheres and melanoma cells with a microscope objective. Using a confocal imaging-based method to reconstruct and analyze the 3D structure, they demonstrated that genetic modifications in these cells can induce morphological changes, and the modified cells can be used as an experimental model to study the correlation between 3D morphology features and diffraction image data. Galanzha et al. [13, 14] introduce in vivo FCM with photoacoustic and photothermal detection schematics. Conventional FCM provides fast multi-parameter quantification of the biological properties of individual cells at subcellular and molecular levels. Nevertheless, the invasive extraction of cells from a living system modifies their properties, preventing their long-term study in their native environment. In vivo FCM of blood and lymph vessels with natural bioflows overcomes these problems. Cells can be detected label-free, by endogenous chromophores and pigments or by advanced strongly absorbing nanoparticles as novel labels. Both approaches are demonstrated for noninvasive rapid detection and treatment of metastases in lymph flow and sentinel lymph nodes by an integrated system of multicolor photoacoustic lymph FCM/lymphography and photothermal therapy. Bykov et al. [15] employ Doppler OCT imaging of cytoplasm shuttle flow in a slime mold Physarum polycephalum. Radial contractions of the gel-like walls of the strands and the velocity distributions in the sol-like endoplasm streaming along the plasmodial strands are imaged. The motility inhibitor effect of carbon dioxide on the cytoplasm shuttle flow and strand wall contraction is shown.

A single-cell encapsulation method based on a microfluidic multi-step droplet splitting system

<div>Abstract<p>Fluorescent proteins are widely used to study molecular and cellular events, yet this traditionally relies on delivery of excitation light, which can trigger autofluorescence, photoxicity, and photobleaching, impairing their use <i>in vivo</i>. Accordingly, chemiluminescent light sources such as those generated by luciferases have emerged, as they do not require excitation light. However, current luciferase reporters lack the brightness needed to visualize events in deep tissues. We report the creation of chimeric eGFP-NanoLuc (GpNLuc) and LSSmOrange-NanoLuc (OgNLuc) fusion reporter proteins coined LumiFluors, which combine the benefits of eGFP or LSSmOrange fluorescent proteins with the bright, glow-type bioluminescent light generated by an enhanced small luciferase subunit (NanoLuc) of the deep-sea shrimp <i>Oplophorus gracilirostris</i>. The intramolecular bioluminescence resonance energy transfer that occurs between NanoLuc and the fused fluorophore generates the brightest bioluminescent signal known to date, including improved intensity, sensitivity, and durable spectral properties, thereby dramatically reducing image acquisition times and permitting highly sensitive <i>in vivo</i> imaging. Notably, the self-illuminating and bifunctional nature of these LumiFluor reporters enables greatly improved spatiotemporal monitoring of very small numbers of tumor cells via <i>in vivo</i> optical imaging and also allows the isolation and analyses of single cells by flow cytometry. Thus, LumiFluor reporters are inexpensive, robust, noninvasive tools that allow for markedly improved <i>in vivo</i> optical imaging of tumorigenic processes. <i>Cancer Res; 75(23); 5023–33. ©2015 AACR</i>.</p></div>

<div>Abstract<p>Fluorescent proteins are widely used to study molecular and cellular events, yet this traditionally relies on delivery of excitation light, which can trigger autofluorescence, photoxicity, and photobleaching, impairing their use <i>in vivo</i>. Accordingly, chemiluminescent light sources such as those generated by luciferases have emerged, as they do not require excitation light. However, current luciferase reporters lack the brightness needed to visualize events in deep tissues. We report the creation of chimeric eGFP-NanoLuc (GpNLuc) and LSSmOrange-NanoLuc (OgNLuc) fusion reporter proteins coined LumiFluors, which combine the benefits of eGFP or LSSmOrange fluorescent proteins with the bright, glow-type bioluminescent light generated by an enhanced small luciferase subunit (NanoLuc) of the deep-sea shrimp <i>Oplophorus gracilirostris</i>. The intramolecular bioluminescence resonance energy transfer that occurs between NanoLuc and the fused fluorophore generates the brightest bioluminescent signal known to date, including improved intensity, sensitivity, and durable spectral properties, thereby dramatically reducing image acquisition times and permitting highly sensitive <i>in vivo</i> imaging. Notably, the self-illuminating and bifunctional nature of these LumiFluor reporters enables greatly improved spatiotemporal monitoring of very small numbers of tumor cells via <i>in vivo</i> optical imaging and also allows the isolation and analyses of single cells by flow cytometry. Thus, LumiFluor reporters are inexpensive, robust, noninvasive tools that allow for markedly improved <i>in vivo</i> optical imaging of tumorigenic processes. <i>Cancer Res; 75(23); 5023–33. ©2015 AACR</i>.</p></div>

Cell manipulation and single cell characterization on microfluidic devices

Despite recent advances in single-cell analysis techniques, the ability of single-cell analysis platforms to track specific cells that secreted cytokines remains limited. Here, we report a microfluidic droplet-based fluorescence imaging platform that can analyze single cell-secreted vascular endothelial growth factor (VEGF), an important regulator of physiological and pathological angiogenesis, to explore cellular physiological clues at the single-cell level. Two kinds of silica nanoparticle (NP)-based immunoprobes were developed, and they were bioconjugated to the membrane proteins of the probed cell surface via the bridging of secreted VEGF. Thus, an immunosandwich assay was built above the probed cell via fluorescence imaging analysis of each cell in isolated droplets. This analytical platform was used to compare the single-cell VEGF secretion ability of three cell lines (MCF-7, HeLa, and H8), which experimentally demonstrates the cellular heterogeneity of cells in secreting cytokines. The uniqueness of this method is that the single-cell assay is carried out above the cell of interest, and no additional carriers (beads or reporter cells) for capturing analytes are needed, which dramatically improves the availability of microdroplets. This single-cell analytical platform can be applied for determining other secreted cytokines at the single-cell level by changing other immune pairs, which will be an available tool for exploring single-cell metabonomics.

Data generated using single-cell RNA-sequencing has the potential to transform understanding of the cerebral circulation and advance clinical care. However, the high volume of data, sometimes generated and presented without proper pathophysiological context, can be difficult to interpret and integrate into current understanding of the cerebral circulation and its disorders. Furthermore, heterogeneity in the representation of brain regions and vascular segments makes it difficult to compare results across studies. There are currently no standards for tissue collection and processing that allow easy comparisons across studies and analytical platforms. There are no standards either for single-cell data analysis and presentation. This topical review introduces single-cell RNA-sequencing to physicians and scientists in the cerebrovascular field, with the goals of highlighting opportunities and challenges of applying this technology in the cerebrovascular field and discussing key concepts and knowledge gaps that can be addressed by single-cell RNA-sequencing.

Introduction: Tumor-associated macrophages (TAMs) constitute a significant population of immune cells in the tumor microenvironment (TME), mainly exhibiting an anti-inflammatory M2-like phenotype. They secrete angiogenic growth factors that support tumor growth with an immunosuppressive TME and contribute to developing resistance against immunotherapies. Therefore, there is an increasing effort to switch TAMs from protumoral M2-like to antitumoral and pro-inflammatory M1-like phenotype. Aging is a critical risk factor, with global cancer incidences increasing from 45 to 75 years of age and declining thereafter. The reason behind this decline beyond 75 years of age is unknown but needs further investigation. With the underrepresentation of aged population in preclinical and clinical studies, the role of aging in cancer development and the TME are understudied. Considering the multidimensional role of macrophages in the TME, this study determines the effects of age-associated changes in macrophage function and phenotype on tumor development. Objective: To study the changes in macrophage biology with aging and understand the implications of aging macrophages on the TME in murine cancer models. Methods: Wildtype mice of ages 6-8 WKS, 3-6 MO, 10-12 MO, and 20-24 MO were implanted with tumor cells (SM1 melanoma, 4T1 breast cancer) to study tumor growth kinetics. Immune cell profiling of TME, including TAMs, was performed using flow cytometry, single-cell RNAseq, and secretome analysis. Bone marrow-derived macrophages isolated from tumor-bearing and healthy mice of the above four age groups were polarized to M1 and M2 phenotypes to study age- and cancer-associated changes in macrophage function, phenotype, and metabolic signature. The changes in macrophage cytokine and chemokine secretion levels with aging were determined by multiplex ELISA assays. Results: Tumor growth in older mice was significantly slower than in younger mice. A distinct change in immune cell populations within the TME and reduced secretion profile of TAMs with aging was observed from single-cell analyses. Additionally, the single-cell secretome analysis reflected an inefficient monocyte-to-macrophage transition in tumors of older mice. The defect in macrophage polarization to M1- and M2-like phenotypes with aging complemented with the sub-optimal function (phagocytosis, antigen presentation, and migration) and metabolic signatures of aged macrophages. Conclusions: The phenotypic, functional, and metabolic changes in macrophages with aging significantly affect tumor growth in murine cancer models. Our study further emphasizes the important role of macrophages in the TME and the need for better representation of aged populations in preclinical and clinical studies. Citation Format: Manasa Suresh, Marie Durr, Xintang Li, Bryan Weselman, Francisco Tapia Belmonte, Nithya Gajendran, David Quiceno-Torres, Sonia Sebaoui, Adhithi Rajesh, Matias Hepp, Alexis Salas-Burgos, Satish Noonepalle, Alejandro Villagra. Changes in macrophage phenotype and function with aging affects tumor growth and tumor microenvironment in murine cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6139.

Traceable impedance-based single-cell pipetting, from a research set-up to a robust and fast automated robot: DispenCell-S1.

This study describes an efficient immune profiling method by FACS-sorting single cells in a 96-well plate to assess clonotype repertoire diversity, paired-chain information, and cell phenotypes in a single, multiplex RT-PCR-based assay without the use of any specialized equipment. It is a cost-effective alternative to conventional single-cell technologies such as microwell arrays or droplet microfluidics. The 96-well plate contains primers for either T-cell receptor (TCR) / or TCR / chains, together with primers for 30 key T-cell markers. T cells from PBMCs are sorted in a single-cell-per-well format. We conduct RT-PCR library prep using the DriverMapTM Adaptive Immune Receptor (AIR) Repertoire Profiling Assay and sequence the CDR3 region. The results show clonotype frequencies, chain pairing details for / and / chains, and T-cell subtype classifications based on gene expression profiling. This enables in-depth analysis of TCR gene rearrangements at the single-cell level, enhancing understanding of T cell development, proliferation, and clonality, which are important in studying cancer, immunodeficiencies, and autoimmune disorders. Additionally, we can effectively identify and characterize γδ T cells by integrating single-cell TCR sequencing with RNA sequencing data, potentially useful in the development of γδ cancer immunotherapies. Overall, this method provides an efficient way to simultaneously analyze clonotypes and immunophenotypes in a single assay.

Microfluidic-based single cells analysis has been of great interest in recent years, promising disease diagnosis and personalized medicine. Current technologies are challenging in bioselectively isolating specific single cells from complex matrices. Herein, a novel microfluidic platform integrated with cell-recognizable aptamer-encoded microwells was specifically developed to isolate single tumor cells with satisfied single-cell occupancy and unique bioselectivity. In this work, the designed microwell-structures enable us to encourage strong 3D local topographic interactions of the target cell surface with biomolecules and regulate the single-cell resolution. Under the optimized size of microwells, the single-cell occupancy was significantly enhanced from 0.5% to 88.2% through the introduction of the aptamer. Analysis of the target cells was directly performed in short time periods (<5.0 min) with small volumes (4.5 μL). Importantly, such an aptamer-enabled microfluidic device shows an excellent selectivity for target single cells isolation compared with three control cells. Subsequently, targeted isolation and analysis of single tumor cells were demonstrated by using artificial complex cell samples at simulated conditions, and various cellular carboxylesterases were studied by time-course measurements of cellular fluorescence kinetics at individual-cell level. Thus, our technique will open up a new opportunity in single-cell level-based disease diagnosis and personalize medicine screening.

Deconstructing stem cell population heterogeneity: Single-cell analysis and modeling approaches