Inkjet Printing Based Separation of Mammalian Cells by Capillary

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Inkjet printing based separation of mammalian cells by capillary electrophoresis Weifei Zhang, Nan Li, Hulie Zeng, Hizuru Nakajima, Jin-Ming Lin, and Katsumi Uchiyama Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02624 • Publication Date (Web): 12 Aug 2017 Downloaded from http://pubs.acs.org on August 13, 2017

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Analytical Chemistry

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Inkjet printing based separation of mammalian cells by capillary electrophoresis

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Weifei Zhang,† Nan Li,‡ Hulie Zeng,† Hizuru Nakajima,† Jin-Ming Lin,‡,* and Katsumi Uchiyama,†,*

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Department of Applied Chemistry, Graduate School of Urban Environmental Sciences, Tokyo Metropolitan University, Minamiohsawa, Hachioji, Tokyo 192-0397, Japan

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Supporting Information Placeholder

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‡

Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, The Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, China

ABSTRACT: This study describes a method to investigate the separation of cells by capillary electrophoresis (CE) coupled with inkjet printing system. The results validated the feasibility of inkjet printing for mammalian cells to achieve the drop-on-demand and convenient sampling into capillary then zone electrophoresis was applied to separate different cells according to their electrophoretic mobility, finally the peak signal were measured by UV detector. Linear relationship between the peak area and the droplet number was obtained within 2 the range of 25~400 drops (R = 0.996) at a fixed cell 6 concentration 10 /mL, indicating that this system could be used for rapid and accurate quantification of cells.

Cell separation is an essential preparatory step in many biological and medical assays. Much works so far have focused on the development of cell separation 1-4 techniques. Cell samples are highly complex, containing many different species at widely different abundance levels. Moreover, the natural variation of cells (even from the same lineage) sometimes confounds the study itself. As a result, it is also crucial to perform fundamental studies to understand different cell type and their property. Thus, development of rapid cell separation with reliable and controllable techniques is important to the studies on cell biology. Up to now, capillary electrophoresis (CE) has been one of the most powerful tools in the separation and analysis of biological particles, such as sub-cellular or5-8 9,10 1 ganisms, bacteria, and entire mammalian cells, because it can analyze sample volume ranging from nano-

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liters to picoliters with fast, efficient separation and easily couple to sensitive detectors. The whole cells (~20μm in diameter) can be separated detected by injecting the intact cell into a capillary with the optimization of all CE steps, including cell sample injection. In conventional injection methods, the whole cells are infused into the capillary via a potential across the capillary (electrokinetic) or a different pressure between inlet and outlet of the 1,11,12 capillary (siphoning). The injection process is often tiring and complex because most work involve manual adjustments and it's not easy to accurately control the volume of sample. Therefore, development of new sam13 ple injection techniques is desired. Inkjet printers are capable of printing at high resolution by ejecting extremely small ink drop. The technology has aroused by increasing interest in biomedical micro-fabrication, since it offers a practical and efficient 14-21 method to dispense biological and material elements, The volume of droplet ejected from an inkjet printer ranges from pL to nL by controlling the condition of inkjet system, according to the requirement of experiment. In recent years, a number of research groups have developed inkjet printing as a technique for the sampling and ejecting cells. In the work by Chen et al., an inkjet printing was developed as a tool for the preparation and analysis of single-cell by probe electrospray ionization 22 mass spectrometry (PESI-MS). Liberski and coworkers demonstrated inkjet was particularly well-suited as a dispensing technique for delivering single living cells to 23 predetermined locations. Zhang et al. reported that the application of inkjet printing was a precise and convenient means for microscale cell to patterning in microfluidic chip. They used the technique for cell co-culture in the

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Figure 1. Set up of cell analysis system with capillary electrophoresis. (a) Illustration of home-made equipment. (b) Illustration of inkjet sampling technique with various concentrations of cells. (c) Principle of cells separation in capillary electrophoresis. microchip and shown that the device could be used to 24 perform drug metabolism and diffusion experiments. Lin and Uchiyama have jointly established a new platform as simple and high reproducibility sample introduction system using inkjet technology to get a wide range of information from single or several cells levels in 25 MALDI-MS analysis. In this work, we present the potential of inkjet injecting as sampling technique for cell separation by capillary electrophoresis combining UV detection for the first time. Through injecting cell suspension, droplets containing cells were generated and precisely introduced into a capillary, and then an immediate separation under a high-voltage electric field was executed. We optimized injection conditions and the pH of running buffer to reduce the separation time and improve the accuracy and sensitivity of our method. The normal and apoptotic cells were resolved owing to the migration rate differences. The analytical assay was also validated by separating three different cell lines, i.e., HUVEC, HepG2 and Caco-2 cells. The sampling method described here presents an easy and reliable way for quantitative cell injection. The design of the platform is shown in Figure 1a. It consists of inkjet sampling system, capillary electrophoresis system, and UV detector. The fixed cells with various concentrations (from high to low) were introduced into the capillary by inkjet system (Figure 1b). The apoptotic and normal cells can be separated by capillary electrophoresis because charge effects migration rate (Figure 1c). The amount of introduced cell was related to cell concentration and the number of droplets. The volume of each droplet was controlled by inkjet system conditions (driving voltage and pulse width). After optimization, driving voltage of this inkjet system was defined at 28 V and the pulse width was 20 μs. In this condition, the droplet could be directly injected into the capillary and volume of a single droplet was 198 pL. The plug length for the single-droplet was 45 µm in the diameter of a 75 μm capillary. (Showing Figure S1, Supporting Information). In order to keep the amount of cell introduced

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uniform and sufficient, the cell concentration and the number of droplets were also optimized. To evaluate the quantification ability of the inkjet sampling system, droplets containing cells were ejected onto glass slides and covered by glycerin to prevent evaporation, then the number of cells was counted under a microscope. The results are shown in Figure 2. The number in the upper right corner of each image represented the number of droplets ejected on the glass slide. The amount of cells was proportional to the number of droplets at a concen6 tration of 1.0 x 10 cells/mL. (Showing Figure S2, Supporting Information). In addition, we confirmed that our method can achieve single cell sampling by optimizing the cell concentration (Figure 2).

Figure 2. Images of the amounts of whole cells ejected on a glass slide with various droplet numbers at two different concentrations. The number shown in each picture shows the droplet number ejected. The pH of the running buffer (50 mM borate buffer containing 6% sucrose) affected the migration time, peak height and the separation of the cells because of its influence on the zeta-potential, the electroosmotic flow (EOF) as well as the surface charge of the cells. In this experiment, the anode was placed at the injection end and the cathode was put at the detector end. The direction of EOF was from the anode to the cathode, while the electric field caused cell to migrate to the opposite direction because of their negative charge.


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Figure 3. (a) Relationship between EOF and pH. (b) Effect of buffer pH on migration time (I) and current (II). The experimental conditions are outlined in the experimental section.

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experimental conditions, the electrophoretic mobility of -4 -2 -1 -1 HepG2 cells was found to be - 1.967 x 10 cm s v . On the basis of the above described investigation, 50 mM borate buffer (pH 7.4) containing 6% sucrose which is used to increase the viscosity of buffer and suppress the precipitation of cell, 10 kV for separation voltage were adopted as appropriate analytical conditions for the quantitative assessment of our system. The electropherogram of various amounts of HepG2 cells was shown in Figure 4b. The peak height of HepG2 cells increased with the increasing of the number of droplets. Unlike small molecule, cell samples have obviously electrophoretic heterogeneity owing to the heterogeneity of size, surface shape and charge property. Even the same kind of cell has subtle heterogeneity. Besides, some uncontrollable factors change with the experiment condition and times. As a result, it's not easy to keep the retention time completely the same in every time. Figure 4b the inset shows the calibration curve obtained by peak area vs droplet number. It was found that the curve was linear in the range of 25 - 400 droplet number, and the linear relation2 ship was obtained (R = 0.996) and the fitting formula was Y = 0.089x - 0.932 (where Y is peak area and X is the droplet number). The excellent linearity indicated that inkjet system coupled with CE shows that the method enables quantitative cell analysis, which can achieve the excellent reproducibility, rapidity, simplicity, and accuracy.

The fact that cell migrated from injection end to the detector end shows that EOF to is larger than the electrophoretic mobility of the cell. In Figure 3a, we used neutral molecule, acetone, to calculate the EOF in borate buffer at different pH condition. The EOF increase of pH from 4.5 to 7.5 corresponding to the dissociation of the silanol group on the surface of capillary. In Figure 3b, we used HepG2 cell to observe the migration time in different pH values (4.5 5.5 6.5 7.4 and 8.5). The results showed that the peak current increased concomitant with the increasing of pH but migration time decreased. According to the results, we selected the running buffer pH of 7.4 in the following experiments, because in this condition, the EOF was the largest and the migration rate of cells was fastest, besides, the influence to the surface charge of cells were the smallest owing to the similar pH in vivo environment. Under the optimized condition, it was found that the retention time of HepG2 cells was in 4.372 min, as shown in Figure 4a. Cell mobility in the capillary resulted from a combination of EOF and its own electrophoretic mobility. The CZE mode was determined by EOF and cell mobility. The VE can easily be calculated as VE = V - VOS Where V and VOS are the velocities of apparent migration of the cells and the electroosmotic migration rate by EOF, respectively. VOS in optimal condition was meas-1 ured with acetone as neutral material to be 0.1882 cm s based upon the results shown in Figure S3. Under our

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Figure 4. (a) CE electropherograms of cells from HepG2 cell. (b) CE pattern of HepG2 cells with droplet various number 25 ~ 400. The inset shows the calibration curve of HepG2 cells. Effluent was detected at 280 nm. Buffer: 50 mM borate containing 6% sucrose; and the electrophoretic field was 182 V/cm.HepG2Cell at the concentra6 tion of 1 x 10 cells/mL.



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To evaluate the accuracy of the established method, normal and apoptosis HepG2 cells were mixed, we successfully realized the separation of the mixed cell suspension by capillary electrophoresis (Figure 5a). The electrophoretic behavior of a cell is directly related to its surface charge, which will change when the lesions occur owing to the variation of cell surface. The results shows that the surface charge of apoptosis cells was lower than that of the normal cells, which are in accordance with 26,27 the previous reports. Therefore, investigating the electrophoretic behavior of a cell contributes to the evaluation of the injury severity and will even predict the 27 occurrence of diseases. Further, we separated different kinds of cell, the difference of migration rate between them was caused by the variation in size and surface charge. The retention times of three cells was shown in Figure S4. Typical CZE patterns of cells from HUVEC, HepG2 and Caco-2 are shown in Figure 5b. When high voltage was applied, EOF exerted an equal effect on different cells. Therefore, the retention time was related to the density of cell surface charge. The higher the density of the negative charge, the slower the cell migrated. The obtained electrophoretic patterns indicate that the density of the negative charge of Caco-2 cells was the lowest, and the density of the negative charge of HUVEC cells

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was the highest among the three. It should be noted, in Figure 5b, the height of different peak 1, 2, 3 did not reflect the quantitative relationship to the corresponding cells, because of the heterogeneity in these three types of cells, such as size, surface shape and charge property. In summary, this work introduces the method using CZE in combination with inkjet sampling for quantitative cell separation. Compared with conventional sampling technique, the inkjet sampling can be precisely manipulated with spatial and temporal control. Importantly, it can realize quantitative analysis of cell by adjusting the cell concentration and the number of droplets. What's more, we can easily achieve the single cell sampling with inkjet, in our future work, we will perform the analysis of single cell by developing more sensitive detectors.

272 ASSOCIATED CONTENT 273 Supporting Information 274 The Supporting Information is available free of 275 charge on the ACS Publications website: Experi276 mental description; figures showing Optimization 277 of the inkjet driving wave form; and supplementary 278 figures. 279 AUTHOR INFORMATION 280 Corresponding Author 281 *E-mail: [email protected] 282 *E-mail: [email protected] 283 284 Notes 285 The authors declare no competing financial 286 ests.

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287 ACKNOWLEDGMENT

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Figure 5. The determination of different property of cell by CE. (a) Electropherogram for normal and apoptosis HepG2 cells. Peak (1) apoptosis cells (2) normal cells. (b) Electropherogram of a mixture of three kind of cells. (1) Caco-2 cells (2) HepG2 cells (3) HUVEC cells. Cell at the 6 concentration of 1 x 10 cells/mL (200 droplets). The separation conditions are outlined in the experimental section.

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This work was financially supported by High technology research funding by Tokyo Metropolitan University and National Natural Science Foundation of China (Nos. 81373373, 21435002, 21621003).

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Inkjet Printing Based Separation of Mammalian Cells by Capillary

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