Advance of microfluidic flow cytometry enabling high-throughput characterization of single-cell electrical and structural properties.

This paper reported a micro flow cytometer capable of high-throughput characterization of single-cell electrical and structural features based on constrictional microchannels and deep neural networks. When single cells traveled through microchannels with constricted cross-sectional areas, they effectively blocked concentrated electric field lines, producing large impedance variations. Meanwhile, the traveling cells were confined within the cross-sectional areas of the constrictional microchannels, enabling the capture of high-quality images without losing focuses. Then single-cell features from impedance profiles and optical images were extracted from customized recurrent and convolution networks (RNN and CNN), which were further fused for cell-type classification based on support vector machines (SVM). As a demonstration, two leukemia cell lines (e.g., HL60 vs. Jurkat) were analyzed, producing high-classification accuracies of 99.3% based on electrical features extracted from Long Short-Term Memory (LSTM) of RNN, 96.7% based on structural features extracted from Resnet18 of CNN and 100.0% based on combined features enabled by SVM. The microfluidic flow cytometry developed in this study may provide a new perspective for the field of single-cell analysis.

Development of a Microfluidic Flow Cytometer with a Uniform Optical Field (Uni-µFCM) Enabling Quantitative Analysis of Single-Cell Proteins and Its Applications in Leukemia Gating, Tumor Classification, and Hierarchy of Cancer Stem Cells.

Fast and quantitative estimation of single-cell proteins with various distribution patterns remains a technical challenge. Here, a microfluidic flow cytometer with a uniform optical field (Uni-µFCM) was developed, which enabled the translation of multicolor fluorescence signals of bound antibodies into targeted protein numbers with arbitrary distributions of biological cells. As the core of Uni-µFCM, a uniform optical field for optical excitation and fluorescence detection was realized by adopting a microfabricated metal window to shape the optical beam for excitation, which was modeled and validated by both numerical simulation and experimental characterization. After the validation of Uni-µFCM in single-cell protein quantification by measuring single-cell expressions of three transcriptional factors from four cell lines of variable sizes and origins, Uni-µFCM was applied to (1) quantify membrane and cytoplasmic markers of myeloid and lymphocytic leukocytes to classify cell lines and normal and patient blood samples; (2) measure single-cell expressions of key cytokines affiliated with gene stabilities, differentiating paired oral and colon tumor cell lines with varied malignancies, and (3) quantify single-cell stemming markers of liver tumor cell lines, cell subtypes, and liver patient samples to determine a variety of lineage hierarchy. By quantitatively assessing complex cellular phenotypes, Uni-µFCM substantially expanded the phenotypic space accessible to single-cell applications in leukemia gating, tumor classification, and hierarchy determination of cancer stem cells.

Crossing constriction channel-based microfluidic cytometry capable of electrically phenotyping large populations of single cells.

This paper presents a crossing constriction channel-based microfluidic system for high-throughput characterization of specific membrane capacitance (Csm) and cytoplasm conductivity (σcy) of single cells. In operations, cells in suspension were forced through the major constriction channel and instead of invading the side constriction channel, they effectively sealed the side constriction channel, which led to variations in impedance data. Based on an equivalent circuit model, these raw impedance data were translated into Csm and σcy. As a demonstration, the developed microfluidic system quantified Csm (3.01 ± 0.92 µF cm-2) and σcy (0.36 ± 0.08 S m-1) of 100 000 A549 cells, which could generate reliable results by properly controlling cell positions during their traveling in the crossing constriction channels. Furthermore, the developed microfluidic impedance cytometry was used to distinguish paired low- and high-metastatic carcinoma cell types of SACC-83 (ncell = ∼100 000) and SACC-LM cells (ncell = ∼100 000), distinguishing significant differences in both Csm (3.16 ± 0.90 vs. 2.79 ± 0.67 µF cm-2) and σcy (0.36 ± 0.06 vs.0.41 ± 0.08 S m-1). As high-throughput microfluidic impedance cytometry, this technique may add a new marker-free dimension to flow cytometry in single-cell analysis.

A microfabricated 96-well wound-healing assay.

This article presents a microfabricated 96-well wound-healing assay enabling high-throughput measurement of cellular migration capabilities. Within each well, the middle area is the wound region, made of microfabricated gold surface with self-assembled PEG repellent for cell seeding. After the formation of a cellular confluent monolayer around the wound region, collagen solution was applied to form three-dimensional matrix to cover the PEG surface, initiating the wound-healing process. By interpreting the numbers of migrated cells into the wound regions as a function of specific stimuli with different concentrations, EC50 (half-maximal effective concentration) was obtained. Using H1299 as a model, values of EC50 were quantified as 8% and 160 ng/ml for fetal bovine serum and CXCL12, respectively. In addition, the values of EC50 were demonstrated not to be affected by variations in compositions of extracellular matrix and geometries of wounds, which can thus be regarded as an intrinsic marker. Furthermore, the migration capabilities of a second cell type (HeLa) were characterized by the developed wound-healing assay, producing EC50 of 2% when fetal bovine serum was used as the stimuli. These results validated the proposed high-throughput wound-healing assay, which may function as an enabling tool in studying cellular capabilities of migration and invasion. © 2017 International Society for Advancement of Cytometry.

A Microfabricated 96-Well 3D Assay Enabling High-Throughput Quantification of Cellular Invasion Capabilities.

This paper presents a 96-well microfabricated assay to study three-dimensional (3D) invasion of tumor cells. A 3D cluster of tumor cells was first generated within each well by seeding cells onto a micro-patterned surface consisting of a central fibronectin-coated area that promotes cellular attachment, surrounded by a poly ethylene glycol (PEG) coated area that is resistant to cellular attachment. Following the formation of the 3D cell clusters, a 3D collagen extracellular matrix was formed in each well by thermal-triggered gelation. Invasion of the tumor cells into the extracellular matrix was subsequently initiated and monitored. Two modes of cellular infiltration were observed: A549 cells invaded into the extracellular matrix following the surfaces previously coated with PEG molecules in a pseudo-2D manner, while H1299 cells invaded into the extracellular matrix in a truly 3D manner including multiple directions. Based on the processing of 2D microscopic images, a key parameter, namely, equivalent invasion distance (the area of invaded cells divided by the circumference of the initial cell cluster) was obtained to quantify migration capabilities of these two cell types. These results validate the feasibility of the proposed platform, which may function as a high-throughput 3D cellular invasion assay.

A Tubing-Free Microfluidic Wound Healing Assay Enabling the Quantification of Vascular Smooth Muscle Cell Migration.

This paper presents a tubing-free microfluidic wound healing assay to quantify the migration of vascular smooth muscle cells (VSMCs), where gravity was used to generate a laminar flow within microfluidic channels, enabling cell seeding, culture, and wound generation. As the first systemic study to quantify the migration of VSMCs within microfluidic environments, the effects of channel geometries, surface modifications and chemokines on cellular migration were investigated, revealing that 1) height of the micro channels had a significant impact on cell migration; 2) the surface coating of collagen induced more migration of VSMCs than fibronectin coated surfaces and 3) platelet derived growth factor resulted in maximal cell migration compared to tumor necrosis factor alpha and fetal bovine serum. Furthermore, migrations of five types of VSMCs (e.g., the human vascular smooth muscle cell line, two types of primary vascular smooth cells, and VSMCs isolated from two human samples) were quantified, finding that VSMCs from the cell line and human samples demonstrated comparable migration distances, which were significantly lower than the migration distances of two primary cell types. As a platform technology, this wound healing assay may function as a new model to study migration of VSMCs within microfluidic environments.