Laborless, Automated Microfluidic Tandem Cell Processor for Visualizing Intracellular Molecules of Mammalian Cells

Abstract

Visualization and quantification of intracellular molecules of mammalian cells are crucial steps in clinical diagnosis, drug development, and basic biological research. However, conventional methods rely mostly on labor-intensive, centrifugation-based manual operations for exchanging the cell carrier medium and have limited reproducibility and recovery efficiency.Here we present a microfluidic cell processor that can perform four-step exchange of carrier medium, simply by introducing a cell suspension and fluid reagents into the device. The reaction time period for each reaction step, including fixation, membrane permeabilization, and staining, was tunable in the range of 2 to 15 min by adjusting the volume of the reaction tube connecting the neighboring exchanger modules. We double-stained the cell nucleus and cytoskeleton (F-actin) using the presented device with an overall reaction period of ∼30 min, achieving a high recovery ratio and high staining efficiency. Additionally, intracellular cytokine (IL-2) was visualized for T cells to demonstrate the feasibility of the device as a pretreatment system for downstream flow-cytometric analysis. The presented approach would facilitate the development of laborless, automated microfluidic systems that integrate cell processing and analysis operations and would pave a new path to high-throughput biological experiments.

Multiplex Profiling of Biomarker and Drug Uptake in Single Cells Using Microfluidic Flow Cytometry and Mass Spectrometry

“…This observation is in agreement with the result of particle-tracing simulation as illustrated in Figure C. While centrifugation has traditionally been considered the most common method for washing samples, the centrifugation process has been known to cause sample loss as well as reduced sample viability . To verify this, we performed multiple centrifugations on the cells.…”

“…29 However, during flow, specific binding of recognition-ligands on cells may be adversely affected by larger shearing stress at high flow rate, 30 and massive cell sampling may result in cell loss due to the centrifugal operation employed in sample purification. 31 Thus, integration of a microfluidic chip with MS to conduct simultaneous sample preprocessing and detection under low flow rates will yield better single-cell information so as to enable more effective and comprehensive multiplex profiling of cell phenotype and heterogeneity.…”

Dual-Multivalent-Aptamer-Conjugated Nanoprobes for Superefficient Discerning of Single Circulating Tumor Cells in a Microfluidic Chip with Inductively Coupled Plasma Mass Spectrometry Detection

“…26 Larger-sized CTCs conjugated with sea urchin-DMA-AuNP nanoprobes were sorted in a purifier through a transfer channel (TC), while other smaller-sized cells or particles were directed to outlets 1 and 2 for disposal. By following the principle of HDF, 23,24 the rinsing solution imported from inlet 2 was employed for further purification of the CTCs from the TC channel. The unbound sea urchin-DMA-AuNP nanoprobes and some vesicles were forced to a thin flow region (inner surface of 104 side channels) and were discharged from outlet 4.…”

“…By direct sampling, however, it is highly probable that a single spike is produced, involving multiple CTC events. In this respect, single-cell microfluidic platforms play a significant role in accurate cell alignment and high-resolution single-cell sampling. , Particularly in passive approaches, by relying on Dean drag force in spiral channels, the tumor cells may be actuated in equilibrium positions and separated from complex biological matrices. , In contrast, the initiative protocols based on extra optics, acoustics, or electrochemistry may affect the original metabolism of the cellular surface. , Although some passive spiral microfluidic chips were fabricated for sorting single CTCs with element encoding, other cells/debris with high abundance are million-fold more abundant than CTCs in biological systems, which are most probably subject to potential interference from mistaken encoding; , hence, a series of size-dictated separation chips were developed for replacing medium of target cells based on deterministic lateral displacement (DLD) ,, and hydrodynamic filtration (HDF). , For DLD chips, a large cell separation area and the use of tens of thousands of micropillars make the system more complicated. HDF chips are easier to design and construct, while their potentials in single-cell sorting remain largely unexploited.…”

“…18,19 Although some passive spiral microfluidic chips were fabricated for sorting single CTCs with element encoding, other cells/debris with high abundance are millionfold more abundant than CTCs in biological systems, which are most probably subject to potential interference from mistaken encoding; 9,20 hence, a series of size-dictated separation chips were developed for replacing medium of target cells based on deterministic lateral displacement (DLD) 8,21,22 and hydrodynamic filtration (HDF). 23,24 For DLD chips, a large cell separation area and the use of tens of thousands of micropillars make the system more complicated. HDF chips are easier to design and construct, while their potentials in single-cell sorting remain largely unexploited.…”