Beiyuan Fan

Development of Droplet Microfluidics Enabling High-Throughput Single-Cell Analysis

This article reviews recent developments in droplet microfluidics enabling high-throughput single-cell analysis. Five key aspects in this field are included in this review: (1) prototype demonstration of single-cell encapsulation in microfluidic droplets; (2) technical improvements of single-cell encapsulation in microfluidic droplets; (3) microfluidic droplets enabling single-cell proteomic analysis; (4) microfluidic droplets enabling single-cell genomic analysis; and (5) integrated microfluidic droplet systems enabling single-cell screening. We examine the advantages and limitations of each technique and discuss future research opportunities by focusing on key performances of throughput, multifunctionality, and absolute quantification.

Development of Microfluidic Systems Enabling High-Throughput Single-Cell Protein Characterization

This article reviews recent developments in microfluidic systems enabling high-throughput characterization of single-cell proteins. Four key perspectives of microfluidic platforms are included in this review: (1) microfluidic fluorescent flow cytometry; (2) droplet based microfluidic flow cytometry; (3) large-array micro wells (microengraving); and (4) large-array micro chambers (barcode microchips). We examine the advantages and limitations of each technique and discuss future research opportunities by focusing on three key performance parameters (absolute quantification, sensitivity, and throughput).

Electrical Property Characterization of Neural Stem Cells in Differentiation.

Electrical property characterization of stem cells could be utilized as a potential label-free biophysical approach to evaluate the differentiation process. However, there has been a lack of technology or tools that can quantify the intrinsic cellular electrical markers (e.g., specific membrane capacitance (Cspecific membrane) and cytoplasm conductivity (σcytoplasm)) for a large amount of stem cells or differentiated cells. In this paper, a microfluidic platform enabling the high-throughput quantification of Cspecific membrane and σcytoplasm from hundreds of single neural stem cells undergoing differentiation was developed to explore the feasibility to characterize the neural stem cell differentiation process without biochemical staining. Experimental quantification using biochemical markers (e.g., Nestin, Tubulin and GFAP) of neural stem cells confirmed the initiation of the differentiation process featured with gradual loss in cellular stemness and increased cell markers for neurons and glial cells. The recorded electrical properties of neural stem cells undergoing differentiation showed distinctive and unique patterns: 1) in the suspension culture before inducing differentiation, a large distribution and difference in σcytoplasm among individual neural stem cells was noticed, which indicated heterogeneity that may result from the nature of suspension culture of neurospheres; and 2) during the differentiation in adhering monolayer culture, significant changes and a large difference in Cspecific membrane were located indicating different expressions of membrane proteins during the differentiation process, and a small distribution difference in σcytoplasm was less significant that indicated the relatively consistent properties of cytoplasm during the culture. In summary, significant differences in Cspecific membrane and σcytoplasm were observed during the neural stem cell differentiation process, which may potentially be used as label-free biophysical markers to monitor this process.

Specific membrane capacitance, cytoplasm conductivity and instantaneous Young’s modulus of single tumour cells

As label-free biomarkers, biophysical properties of cells are widely used for cell type classification. However, intrinsic biophysical markers, e.g., specific membrane capacitance (Cspecific membrane), cytoplasm conductivity (σconductivity) and instantaneous Young’s modulus (Einstantaneous) measured for hundreds of single cells were not yet reported. In this study, single cells in suspension (adherent cells treated with trypsin) were aspirated through a microfluidic constriction channel at 25 °C, and the entry processes and impedance profiles were recorded and translated to Cspecific membrane, σconductivity and Einstantaneous. Cspecific membrane, σconductivity and Einstantaneous of five cell types were quantified as 2.10±0.38 μF cm−2, 0.91±0.15 S m−1 and 5.52±0.95 kPa for H460 cells (ncell=437); 2.52±0.54 μF cm−2, 0.83±0.12 S m−1 and 5.54±1.04 kPa for H446 cells (ncell=410); 2.45±0.57 μF cm−2, 0.99±0.18 S m−1 and 5.16±1.68 kPa for A549 cells (ncell=442); 1.86±0.31 μF cm−2, 1.07±0.18 S m−1 and 3.86±0.81 kPa for 95D cells (ncell=415); 2.03±0.35 μF cm−2, 0.99±0.16 S m−1 and 3.49±0.70 kPa for 95C cells (ncell=290). The database of Cspecific membrane, σconductivity and Einstantaneous may serve as a reference for future studies of cellular biophysical properties.

Osteocyte culture in microfluidic devices

This paper presents a microfluidic device (poly-dimethylsiloxane micro channels bonded with glass slides) enabling culture of MLO-Y4 osteocyte like cells. In this study, on-chip collagen coating, cell seeding and culture, as well as staining were demonstrated in a tubing-free manner where gravity was used as the driving force for liquid transportation. MLO-Y4 cells were cultured in microfluidic channels with and without collagen coating where cellular images in a time sequence were taken and analyzed, confirming the positive effect of collagen coating on phenotype maintaining of MLO-Y4 cells. The proliferating cell nuclear antigen based proliferation assay was used to study cellular proliferation, revealing a higher proliferation rate of MLO-Y4 cells seeded in microfluidic channels without collagen coating compared to the substrates coated with collagen. Furthermore, the effects of channel dimensions (variations in width and height) on the viability of MLO-Y4 cells were explored based on the Calcein-AM and propidium iodide based live/dead assay and the Hoechst 33258 based apoptosis assay, locating the correlation between the decrease in channel width or height and the decrease in cell viability. As a platform technology, this microfluidic device may function as a new cell culture model enabling studies of osteocytes.

Development of Microfluidic Impedance Cytometry Enabling the Quantification of Specific Membrane Capacitance and Cytoplasm Conductivity from 100,000 Single Cells

This paper presents a new microfluidic impedance cytometry with crossing constriction microchannels, enabling the characterization of cellular electrical markers (e.g., specific membrane capacitance (Csm) and cytoplasm conductivity (σcy)) in large cell populations (~ 100,000 cells) at a rate greater than 100 cells/s. Single cells were aspirated continuously through the major constriction channel with a proper sealing of the side constriction channel. An equivalent circuit model was developed and the measured impedance values were translated to Csm and σcy. Neural network was used to classify different cell populations where classification success rates were calculated. To evaluate the developed technique, different tumour cell lines, and the effects of epithelial-mesenchymal transitions on tumour cells were examined. Significant differences in both Csm and σcy were found for H1299 and HeLa cell lines with a classification success rate of 90.9% in combination of the two parameters. Meanwhile, tumour cells A549 showed significant decreases in both Csm and σcy after epithelial-mesenchymal transitions with a classification success rate of 76.5%. As a high-throughput microfluidic impedance cytometry, this technique can add a new marker-free dimension to flow cytometry in single-cell analysis.

Single-Cell Mechanical Properties: Label-Free Biomarkers for Cell Status Evaluation

The mechanical behavior of biological cells is largely determined by their cytoskeletons; abnormal cellular functions can change cytoskeletons, leading to variations in cellular mechanical properties. This chapter begins with a summary of the relationships between cellular mechanical properties and various disease processes and changes in cell states: (1) changes in stiffness of red blood cells in cytoskeletal disorders, such as malaria and sickle cell anemia; (2) increased cell deformability of invasive cancer cells, compared with benign counterparts; (3) increased stiffness of leukocytes in sepsis; and (4) decreased deformability during the stem cell differentiation process. In the following section, we discuss the well-established techniques that are being used to measure the mechanical properties of single cells, including atomic force microscopy and micropipette aspiration. Finally, we describe the microfluidic approaches—including microfluidic constriction channels, microfluidic optical stretchers, and microfluidic hydrodynamic stretchers—that are being developed as next-generation, automated, and high-throughput techniques for characterization of the mechanical properties of single cells. The advantages and limitations of each technique are compared and future research opportunities are highlighted.

A Microfluidic Fluorescent Flow Cytometry Capable of Quantifying Cell Sizes and Numbers of Specific Cytosolic Proteins

This study presents a microfluidics based cytometry capable of characterizing cell sizes and counting numbers of specific cytosolic proteins where cells were first bound by antibodies labelled with fluorescence and then aspirated into a constriction microchannel in which fluorescent levels were measured. These raw fluorescent pulses were further divided into a rising domain, a stable domain and a declining domain. In addition, antibody solutions with labelled fluorescence were aspirated through the constriction microchannel, yielding curves to translate raw fluorescent levels to protein concentrations. By using key parameters of three domains as well as the calibration curves, cell diameters and the absolute number of β-actins at the single-cell level were quantified as 14.2 ± 1.7 μm and 9.62 ± 4.29 × 105 (A549, ncell = 14 242), 13.0 ± 2.0 μm and 6.46 ± 3.34 × 105 (Hep G2, ncell = 35 932), 13.8 ± 1.9 μm and 1.58 ± 0.90 × 106 (MCF 10 A, ncell = 16 650), and 12.7 ± 1.5 μm and 1.09 ± 0.49 × 106 (HeLa, ncell = 26 246). This platform could be further adopted to measure numbers of various cytosolic proteins, providing key insights in proteomics at the single-cell level.

Single-Cell Protein Assays: A Review

Quantification of single-cell proteomics provides key insights in the field of cellular heterogeneity. This chapter discusses the emerging techniques that are being used to measure the protein copy numbers at the single-cell level, which includes flow cytometry, mass cytometry, droplet cytometry, microengraving, and single-cell barcoding microchip. The advantages and limitations of each technique are compared, and future research opportunities are highlighted.

Absolute Copy Numbers of β-Actin Proteins Collected from 10,000 Single Cells

Semi-quantitative studies have located varied expressions of β-actin proteins at the population level, questioning their roles as internal controls in western blots, while the absolute copy numbers of β-actins at the single-cell level are missing. In this study, a polymeric microfluidic flow cytometry was used for single-cell analysis, and the absolute copy numbers of single-cell β-actin proteins were quantified as 9.9 ± 4.6 × 105, 6.8 ± 4.0 × 105 and 11.0 ± 5.5 × 105 per cell for A549 (ncell = 14,754), Hep G2 (ncell = 36,949), and HeLa (ncell = 24,383), respectively. High coefficients of variation (~50%) and high quartile coefficients of dispersion (~30%) were located, indicating significant variations of β-actin proteins within the same cell type. Low p values (≪0.01) and high classification rates based on neural network (~70%) were quantified among A549, Hep G2 and HeLa cells, suggesting expression differences of β-actin proteins among three cell types. In summary, the results reported here indicate significant variations of β-actin proteins within the same cell type from cell to cell, and significant expression differences of β-actin proteins among different cell types, strongly questioning the properties of using β-actin proteins as internal controls in western blots.