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

Yi Zhang ab, Yang Zhao c, Deyong Chen ab, Ke Wang ab, Yuanchen Wei ab, Ying Xu d, Chengjun Huang *c, Junbo Wang *ab and Jian Chen *ab
aState Key Laboratory of Transducer Technology, Institute of Electronics, Chinese Academy of Sciences, Beijing, P.R. China. E-mail: jbwang@mail.ie.ac.cn; chenjian@mail.ie.ac.cn
bUniversity of Chinese Academy of Sciences, Beijing, P.R. China
cR&D Center of Healthcare Electronics, Institute of Microelectronics, Chinese Academy of Sciences, Beijing, P.R. China. E-mail: huangchengjun@ime.ac.cn
dDepartment of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Ministry of Education, Shanghai Jiao-Tong University School of Medicine, Shanghai, P.R. China

Received 31st October 2018 , Accepted 24th December 2018

First published on 2nd January 2019


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[thin space (1/6-em)]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[thin space (1/6-em)]000) and SACC-LM cells (ncell = ∼100[thin space (1/6-em)]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.


Introduction

As label-free bioelectrical markers, cellular electrical properties (e.g., membrane capacitance and cytoplasm resistance) have emerged as promising electrical indicators to classify cell types and evaluate cellular status.1–3 Since the variations in cellular electrical properties are closely related to physiological and pathological processes in blood cells,4–6 tumor cells7–10 and stem cells,11,12 the high-throughput quantification of these properties is significant.

Conventional characterization techniques of cellular electrical properties include dielectrophoresis (DEP), electrorotation (ROT), and micro electrical impedance spectroscopy (μEIS).3,13 In DEP, cells are polarized under electric fields and the numbers of cells attached onto the electrodes generating electric fields are quantified as a function of the electrical frequency and translated into intrinsic electrical properties of cells by curve fitting the Clausius-Mossotti factor spectra.14,15 However, DEP can only estimate electrical properties based on populations rather than at the single-cell level.8,9,16–18 In ROT, the rotating speeds of single cells in electric fields are obtained to acquire electrical parameters of single cells,14,19,20 where the time-consuming steps of cell manipulation and positioning compromise this technique in the characterization throughput (e.g., tens of21 or hundreds of7 tumor cells). In μEIS, single cells are trapped between pairs of microfabricated electrodes, where frequency sweeping is conducted to obtain impedance data.13,22 This approach again suffers from the key limitation of low throughput (e.g., tens of tumour10,23,24 or stem cells12).

Due to dimensional comparisons of microfluidics with cells, microfluidic platforms have been widely used for single-cell analysis.25–32 By combining μEIS and flow cytometry, microfluidic impedance cytometry was recently developed for high-throughput characterization of single-cell electrical properties.3,32–34 However, due to the lack of equivalent circuit models, in the majority of previously reported microfluidic impedance cytometry studies, only variations in amplitudes and phases of raw electrical signals were reported, whereas intrinsic bioelectrical markers such as membrane capacitances and cytoplasm resistance could not be obtained.6,11,35–43

To address this issue, a constriction channel design was integrated within microfluidic impedance flow cytometry, in which raw impedance data of cells traveling in the constriction channels were translated into inherent cellular bioelectrical markers such as specific membrane capacitance (Csm) and cytoplasm conductivity (σcy) based on equivalent circuit models.44,45 However, since this technique requires image processing of the cell travelling process in the constriction channel, it still has relatively low throughput and can only report data from hundreds of single cells.44,46,47

To solve this issue, side channels were recently included in the aforementioned constriction channel design to predefine effective membrane areas of travelling cells without image processing, leading to the characterization of Csm and σcy from 100[thin space (1/6-em)]000 single cells.48 However, in this approach, during cell travelling processes in the constriction channels, cells may deform into the side channels, compromising the corresponding electrical models and producing unreliable values of single-cell electrical properties.

To address the aforementioned technical difficulties, this paper presents a crossing constriction channel for single-cell electrical property characterization, where cell positions during their travelling processes in the crossing constriction channels are properly controlled. More specifically, a travelling cell is forced through the major constriction channel and effectively seals the side constriction channel instead of invading it since fluid flow directions in the crossing constriction channels are properly defined. The corresponding impedance variations due to travelling cells are then translated into Csm and σcy based on an equivalent circuit model. In comparison to previous techniques,44,45,48 this new approach enables high-throughput and reliable characterizations of Csm and σcy of single cells.

Materials and methods

Materials

Unless otherwise indicated, all cell-culture reagents were purchased from Life Technologies Corporation (USA). Materials required for device fabrications include SU-8 negative photoresist (MicroChem Corporation, USA), AZ serial positive photoresist (AZ Electronic Materials Corporation, USA) and 184 silicone elastomer (Dow Corning Corporation, USA). Relevant materials used in cell cultures include RPMI-1640 Media (GIBICO, Life Technologies Corporation, USA, Cat: 11875), DMEM Media (GIBICO, Life Technologies Corporation, USA, Cat: 11995), fetal bovine serum (GIBICO, Life Technologies Corporation, USA, Cat: 10099), penicillin/streptomycin (GIBICO, Life Technologies Corporation, USA, Cat: 15140), 0.25% trypsin (GIBICO, Life Technologies Corporation, USA, Cat: 15050), and phosphate buffer saline (GIBICO, Life Technologies Corporation, USA, Cat: C10010500BT).

Cell culture

All cell lines were purchased from China Infrastructure of Cell Line Resources and cultured in a cell incubator (3111, Thermo Scientific, USA) at 37 °C in 5% CO2. More specifically, the lung cancer cell line of A549 was cultured with RPMI-1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Paired salivary adenoid cystic carcinoma cell types of SACC-83 (parental cells) and SACC-LM (lung metastasis cells) were cultured with DMEM media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, respectively. Prior to experiments, cells in logarithmic phases were trypsinized, centrifuged and resuspended in phosphate buffered saline at a concentration of ∼5 million cells per mL.

Device design and fabrication

In this study, a crossing constriction channel was adopted in the microfluidic impedance flow cytometry, which mainly includes a microfluidic device and an impedance analyser (Fig. 1). The working principle is as follows: a travelling cell was forced through the major constriction channel and effectively sealed the side constriction channel rather than invading it since fluid flow directions in the crossing constriction channels were properly defined. Then, the electric lines confined within the side constriction channel were blocked by the travelling cell, leading to impedance variations that were measured by the impedance analyzer and then translated into Csm and σcy based on an equivalent circuit model. Please note that in this equivalent circuit model, the effective membrane area of the travelling cell is defined by the cross-sectional area of the side constriction channel and thus, image processing is not required in this study for the quantification of Csm and σcy.
image file: c8an02100g-f1.tif
Fig. 1 Schematic (a) and the equivalent electrical model (b) of the crossing constriction channel-based microfluidic system for high-throughput characterization of cellular specific membrane capacitance and cytoplasm conductivity. In operations, a travelling cell is forced through the major constriction channel and effectively seals the side constriction channel, leading to variations in impedance data. These raw impedance data are then translated into Csm and σcy based on an equivalent circuit model, where cellular electrical components are represented by Rcy and Cm in series with translating equations included. The crossing constriction channel with predefined fluid flow directions and rates is proposed in this study, where the deformed cell in the major constriction channel can seal rather than invade the side constriction channel, ensuring the feasibility of the equivalent circuit model.

As the key component of this cytometry, the crossing constriction channel consists of (I) a major constriction channel (a cross-sectional area of 9 μm in height and 11 μm in width) for cell travelling and deformation and (II) a side constriction channel (a cross-sectional area of 9 μm in height and 6 μm in width) for impedance measurements (Fig. 1(a)). Please note that the geometries of the major and side constriction channels were derived from previous publications,45,48 where the relative dimensions of cells and cross-sectional areas of constriction channels were primarily considered for determining channel geometries.

Unlike the observation reported in a previous publication,48 the side constriction channel in this study was connected to the surrounding environment (inlets) through high-fluid resistant units. In the case of no travelling cells, the fluid in the side constriction channel flowed toward the crossing point since a negative pressure was applied at the outlet of the major constriction channel. An incoming cell at the crossing point preferred to recover from the deforming shape formed by the major constriction channel and expand into the side constriction channel. The fluid flow direction in the side constriction channel counterbalanced the invading trend of the cell, which sealed the side channel instead of invading it during its travelling process (Fig. 1(a)).

The proper regulation of cell positions in the travelling point of the crossing constriction channel depends on the fluid flow rates in the side constriction channel. If the flow rate is very low, it cannot effectively confine the expanding trends of cells and if the flow rate is very high, it generates high shear stress, which may compromise cell membranes. Thus, high-fluid resistant units were included in the side constriction channel to regulate the flow rates, where two flow resistant ratios between the side and the major constriction channels (e.g., 60[thin space (1/6-em)]:[thin space (1/6-em)]1 and 110[thin space (1/6-em)]:[thin space (1/6-em)]1) were designed in this study for comparison.

Fig. 1(b) shows the equivalent circuit model of a travelling cell in the crossing constriction channel, where cellular electrical components are represented by Rcy and Cm in series.1 In addition, since the travelling cell cannot completely seal the side constriction channel, there exist electric lines passing around the cell under measurement, which is represented by leakage resistor Rleak. Since the travelling cell only seals the side constriction channel instead of invading it, the effective area of Cm is the cross-sectional area of the side constriction channel (Asc),48 enabling the translation of Cm to Csm (Fig. 1(b)). Furthermore, for the cytoplasm portion, Rcy was translated into σcy, leveraging the cross-sectional area of the side constriction channel (Asc), the width of the major constriction channel (Wmc) and a modification factor (Mf) (Fig. 1(b)). The modification factor Mf was used to address the widening of the electric lines within the cytoplasm portion after penetrating the membrane portion.48 The detailed translating process from raw impedance data to Csm and σcy can be found in previous publications.44,45

The device fabrication was modified using a previously reported method,48 which mainly relies on soft lithography to replicate PDMS-based microfluidic devices from a two-layer SU-8 mold. The key fabrication steps are as follows: (I) the first layer of SU-8 to form the crossing constriction channel was spin-coated on a glass substrate, soft baked, exposed and post-baked without development. (II) The second layer of SU-8 to form the cell loading channel was spin-coated on the first layer of SU-8, soft-baked, exposed with alignment, post-baked, developed and hard-baked. (III) PDMS prepolymer and curing agents were mixed, degassed, poured on channel masters and baked in an oven. The fully cured PDMS structures were then peeled from the SU-8 masters with channel inlets and outlets punched through. (IV) The planer gold electrodes were patterned based on the lift-off process. (V) After oxygen plasma treatments, the PDMS layer and the patterned gold layer were bonded together with alignments.

Device operation and data analysis

In operations, the flow channels, inlets and outlets of the crossing constriction channel were first filled with culture medium. Then, a droplet of cell suspension (5 × 106 cells per ml) was pipetted in the inlet of the major constriction channel, and negative pressure of 5 kPa generated from a pressure calibrator (DPI-610 pressure calibrator, Druck, UK) was applied at the outlet of the major constriction channel to aspirate cells continuously through the crossing constriction channel. The inlets of the side constriction channel were left open to the external environment without the application of additional pressure sources, and the pressure source at the outlet of the major constriction channel regulated the fluid flow rates in the side constriction channel.

Furthermore, in experiments, evaporation of cell culture medium was observed by gradual changes in the basal impedance values and thus, cell culture medium was supplemented in the inlets of the crossing constriction channel. Since the drifts of basal impedance values were taken into consideration in the electrical modeling, no effects were observed on the measurement results of cellular electrical properties.

A two-frequency sinusoidal voltage signal (100 kHz + 250 kHz) was generated by a lock-in amplifier (model 7270 DSP lock-in amplifier, Signal Recovery, USA) and applied between the side constriction channels to monitor the impedance profiles of travelling single cells. To monitor the cell travelling status at the crossing constriction channel, a microscope (IX83, Olympus, Japan) in connection with a high-speed camera (M320S, Phantom, USA) at 3000 fps was employed to record images of the cell travelling process and to determine whether the travelling cells can invade into the side constriction channels.

The measured impedance data were translated into Csm and σcy using the proposed equivalent circuit model. Briefly, impedance values without travelling cells were used to calculate the equivalent impedance of the crossing constriction channel. Then, impedance pulses for individual cells at 100 and 250 kHz were processed, where the widths of pulses were translated into “passing times” and the peaking impedance data of the pulses were translated into “filling ratios” (a ratio of the cross-section area of an aspirated cell to the cross-section area of the side constriction channel, which was translated from Rleak in the electrical model), membrane capacitances (Cm) and cytoplasm resistances (Rcy).44,45 Events with passing times lower than 0.1 ms and/or filling ratios lower than 80% were treated as passing cell fragments rather than intact biological cells and thus, they were no longer processed. For the remaining cells, Cm and Rcy were further translated into Csm and σcy. The detailed translating process from raw impedance data to Csm and σcy can be found in previous publications.44,45

Statistics

The measurements of multiple samples were conducted with results expressed by averages and standard deviations. The student t test was used for comparisons, where the values of P < 0.001 (*) were considered as statistically significant.

Results and discussion

The constriction channel design with a cross-section area smaller than that of cells was initially used for quantifying cellular mechanical properties,49–57 where cells were deformed into the constriction channel with their entry behaviours monitored as biophysical markers. Then, this approach was used for studying cellular electrical properties,48,58–60 ATP synthesis61 and delivery of gene vectors.61 However, in previous designs of constriction channels for characterizing cellular electrical properties, the cell positions during the travelling processes through the constriction channels were not properly controlled, leading to inconsistency with the corresponding electrical models and inaccurate measurement results.48

This paper presents a crossing constriction channel design for single-cell electrical property characterization, where cell positions during the travelling processes in the crossing constriction channels are properly controlled. Fig. 2(a) demonstrates time-sequence microscopic images of a travelling A549 cell through the crossing point of the constriction channel, which was shown to squeeze through the major constriction channel (9.7 ± 0.3 μm in height and 11.0 ± 0.2 μm in width); it sealed the side constriction channel (9.7 ± 0.3 μm in height and 6.1 ± 0.4 μm in width) instead of invading it. These results validated the design of this crossing constriction channel, where deformed single cells were confined within the major constriction channel, which were consistent with the results of the equivalent circuit model. These results were derived from the design where the flow resistance ratio of the side and the major constriction channels was 110[thin space (1/6-em)]:[thin space (1/6-em)]1. Since in this design, cells did not invade the side constriction channels, the design with the flow resistance ratio of 60[thin space (1/6-em)]:[thin space (1/6-em)]1 was not tested due to potential concerns that high fluid flow rates in the side constriction channels may compromise the sealing of the side constriction channel by the traveling cell.


image file: c8an02100g-f2.tif
Fig. 2 (a) Time-sequence microscopic images of a travelling cell through the crossing point of the constriction channel, which sealed the side constriction channel instead of invading it due to the fluid flow directions and rates within the side constriction channel. (b) Raw impedance amplitude and phase measurements of A549 cells (a lung tumor cell line) at 100 kHz and 250 kHz, respectively, where amplitude increases and phase decreases for travelling cells at both frequencies. Based on the equivalent circuit model, recorded raw impedance changes were further processed to filling ratio (0.91 ± 0.04) vs. passing time (2.01 ± 18.20 ms) (c), membrane capacitance Cm (2.43 ± 0.75 pF) vs. cytoplasm resistance Rcy (0.33 ± 0.18 MΩ) (d), and specific membrane capacitance Csm (3.01 ± 0.92 μF cm−2) vs. cytoplasm conductivity σcy (0.36 ± 0.08 S m−1) (e), where ncell = ∼100[thin space (1/6-em)]000.

Fig. 2(b) shows raw impedance amplitude and phase measurements of A549 cells at 100 and 250 kHz, respectively, where we observed increase in amplitude and decrease in phase for travelling cells at both frequencies. Furthermore, these raw impedance data were processed to four intermediate parameters including passing time, filling ratio, Rcy and Cm (Fig. 2(c) and (d)). More specifically, passing times were obtained by quantifying the time durations of individual pulse in amplitude and estimated to fall within the range of 0.1–10 ms. Thus, the throughput of this cytometry was estimated to be higher than 100 cells per second. Furthermore, filling ratios were derived from Rleak and an increase in the filling ratio indicated decrease in the leakage currents. The filling ratios in this cytometry mainly fell in the range of 80%–100% and if the filling ratio for a travelling cell was very low (e.g., lower than 80% in this study), it was no longer processed further since it may represent a cell fragment or a broken cell with compromised membrane portions.

For the potential concern of channel blockage, in each experiment, which lasted for one hour after trypsinization of cells, no replacement of constriction channels was conducted due to channel blockage. Thus, for each cell type, impedance data from ∼100[thin space (1/6-em)]000 cells were collected. For individual experimental durations of 5 minutes, one or two instances of channel blockage were noticed. In this scenario, a positive pressure was applied at the outlet of the major constriction channel to push the blocking particles back into the cell loading channel, which were then flushed away in a bypass channel. The detailed design of the bypass channel can be found in a previous publication.48

Based on the equivalent circuit model, Rcy and Cm were further translated into size-independent and intrinsic bioelectrical markers of Csm and σcy (Fig. 2(e)). More specifically, in this study, Csm and σcy of A549 cells were quantified as 3.01 ± 0.92 μF cm−2 and 0.36 ± 0.08 S m−1 (ncell = ∼100[thin space (1/6-em)]000), which were higher than the values of 1.86 ± 0.45 μF cm−2 of Csm and 0.22 ± 0.06 S m−1 of σcy reported in a previous publication.48

In a previous study,48 travelling cells invaded into the side constriction channels, whereas the calculations of cellular electrical properties were based on the assumptions of no cell invasions into the side constriction channels. The used equivalent membrane area of the membrane portion was higher than the actual cross-sectional area of the constriction channel, producing lower Csm than real values. In microfabrications, the cross-sectional areas at the entrance of the side constriction channel were larger than the actual values within the side constriction channel due to optical diffractions in the step of exposure. In addition, the used equivalent length of the cytoplasm portion in electrical property calculation was lower than the actual values, producing lower σcy than real values. In this study, the reported values of Csm and σcy were higher than the values given in a previous study, suggesting that the platform developed in this study can properly define cell positions during their travelling processes in the crossing constriction channels, leading to more reliable characterizations of single-cell electrical properties.

In a second demonstration, crossing constriction channel-based microfluidic impedance cytometry was used to classify paired low- and high-metastatic carcinoma cell types of SACC-83 and SACC-LM cells with different invasion abilities. Fig. 3(a) shows microscopic images of SACC-83 and SACC-LM cells invading into the middle portion of extracellular matrix under 0%, 2% and 50% FBS based on a home-developed 96-well wound-healing assay.62 In comparison to SACC-83 cells, SACC-LM cells invaded into the central portions of the extracellular matrix more rapidly, which was consistent with the fact that SACC-LM cells were isolated from lung metastasis, whereas SACC-83 cells were isolated from in situ tumours.


image file: c8an02100g-f3.tif
Fig. 3 (a) Microscopic images of invaded SACC-83 and SACC-LM cells based on the home-developed large-array invasion chamber, suggesting different migration capabilities of these two paired cell lines collected from in situ tumours and invaded lymph nodes. (b) Scatter plots of Csmvs. σcy for SACC-83 cells (ncell = ∼100[thin space (1/6-em)]000) and SACC-LM cells (ncell = ∼100[thin space (1/6-em)]000), where statistically significant differences (p < 0.001) in 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) were located (c).

For the values of cell electrical properties, compared to SACC-83 cells with Csm and σcy quantified as 3.16 ± 0.90 μF cm−2 and 0.36 ± 0.06 S m−1 (ncell = ∼100[thin space (1/6-em)]000), SACC-LM demonstrated significant changes in intrinsic electrical properties, with decrease in Csm of 2.79 ± 0.67 μF cm−2 and increase in σcy of 0.41 ± 0.08 S m−1 (ncell = ∼100[thin space (1/6-em)]000) (Fig. 3(b) and (c)). Both statistically significant differences in Csm and σcy (p < 0.001) were found, indicating that these electrical properties can be used to distinguish these paired tumour cell types.

As shown in Fig. 3(b), in comparison to A549 cells, SACC-83 and SACC-LM cells demonstrated more significant variations in specific membrane capacitance and cytoplasm conductivity, respectively. For A549 cells, convergences in both specific membrane capacitance and cytoplasm conductivity were noticed, indicating that the variations in intrinsic electrical properties of SACC-83 and SACC-LM cells can result from variations in cells rather than the developed system. Thus, it was speculated by the authors that there are subtypes in SACC-83 and SACC-LM cells, which is common for some cell lines. Since this was not the focus of the article, no further investigations were conducted and only the classification of SACC-83 and SACC-LM cells based on cellular electrical properties was demonstrated.

Conclusion

In summary, crossing constriction channel-based microfluidic cytometry was developed for quantifying single-cell electrical properties, where cell positions during their travelling processes in the crossing constriction channels were properly controlled. As a demonstration, specific membrane capacitance and cytoplasm conductivity of ∼100[thin space (1/6-em)]000 A549 cells were obtained by the developed microfluidic system, which could produce more accurate results in comparison to previous counterparts. Furthermore, the developed microfluidic impedance cytometry was demonstrated to distinguish paired low- and high-metastatic carcinoma cell types, confirming the potential uses of cellular electrical properties for cell type classification.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The authors would like to acknowledge Prof. Yixiang Wang from the Peking University for the gifts of SACC-83 and SACC-LM cell lines. In addition, the authors would like to acknowledge financial supports from the National Natural Science Foundation of China (Grant No. 61431019, 61825107, 61671430), Chinese Academy of Sciences Key Project Targeting Cutting-Edge Scientific Problems (QYZDB-SSW-JSC011), Instrument Development Program, Youth Innovation Promotion Association and Interdisciplinary Innovation Team of Chinese Academy of Sciences, Instrument Development Program of Beijing Municipal Science and Technology Commission (Z181100009518001).

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Footnote

Co-first authors.

This journal is © The Royal Society of Chemistry 2019