Label-Free Separation of Induced Pluripotent Stem Cells with Anti

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Label-Free Separation of Induced Pluripotent Stem Cells with Anti-SSEA-1 Antibody Immobilized Microfluidic Channel Akihisa Otaka, Kazuki Kitagawa, Takahiko Nakaoki, Mitsuhi Hirata, Kyoko Fukazawa, Kazuhiko Ishihara, Atsushi Mahara, and Tetsuji Yamaoka Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b04070 • Publication Date (Web): 16 Jan 2017 Downloaded from http://pubs.acs.org on January 18, 2017

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Label-Free Separation of Induced Pluripotent Stem Cells with Anti-SSEA-1 Antibody Immobilized Microfluidic Channel

Akihisa Otaka,† Kazuki Kitagawa,†,‡ Takahiko Nakaoki,‡ Mitsuhi Hirata,† Kyoko Fukazawa,§ Kazuhiko Ishihara,§ Atsushi Mahara,† and Tetsuji Yamaoka*,†

†

Department of Biomedical Engineering, National Cerebral and Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan ‡

§

Department of Materials Chemistry, Ryukoku University, Seta, Otsu 520-2194, Japan

Department of Materials Engineering, The University of Tokyo, Bunkyo-ku, Tokyo 113-8656, Japan

KEYWORDS: Cell separation, Phospholipid polymer, Stem cells, Microfluidic channel

ACS Paragon Plus Environment

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ABSTRACT

When induced pluripotent stem cell (iPSC) is routinely cultured, the obtained cells are a heterogeneous mixture, including feeder cells and partially differentiated cells. Therefore, a purification process is required to use them in a clinical stage. We described a label-free separation of iPSCs using a microfluidic channel. Antibodies against stage-specific embryonic antigen 1 (SSEA-1) was covalently immobilized on the channel coated with a phospholipid polymer. After injection of the heterogeneous cell suspension containing iPSCs, the velocity of cell movement under a liquid flow condition was measured. The mean velocity of the cell movement was 2.1 mm/sec in the unmodified channel, while that in the channel with the immobilized-antibody was 0.4 mm/sec. The eluted cells were fractionated by eluting time. As a result, the SSEA-1 positive iPSCs were mainly contained in later fractions, and the proportion of iPSCs was increased from 43% to 82% as a comparison with the initial cell suspension. These results indicated that iPSCs were selectively separated by the microfluidic channel. This channel is a promising device for label-free separation of iPSCs based on their pluripotent state.


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INTRODUCTION

Induced pluripotent stem cells (iPSCs) tend to form a heterogeneous population because of stochastic transcription initiation,1,2 reprogramming inefficiency,3 the presence of feeder cells,4,5 and/or involuntarily differentiated derivatives during cell expansion. This heterogeneity leads to a decrease in the controllability of experimental conditions or difficulties in the clinical application of iPSCs. To overcome this problem, many researchers have proposed procedures to separate iPSCs.6–10 Fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting are commonly used techniques, and many membrane molecules have been proposed as markers of pluripotent cells.8,9,11 However, cell labeling is a time- and labor-consuming step and sometimes results in unintended adverse effects on the change of cell phenotype12 or signal transduction.13 Thus, label-free cell separation is desired in stem cell research. Cell rolling behavior resulting from dynamic cell adhesion to a solid surface with immobilized specific affinitive antibodies or receptors have been investigated for label-free cell separation. Previously, we performed mesenchymal stem cell (MSC) enrichment using a “cell rolling column (CRC),” a column with an immobilized anti-CD34 antibody.14,15 This CRC system works as a continuous cell immunophenotyping column because the density of the marker expressed on the cell membrane reduces cell rolling velocity at an equilibrium of the stress of sweep fluid flow and transient tether formation between the membrane antigen and column antibody.16 Likewise, other research groups also reported that cell rolling behavior on microfluidic channels coated with E- or P-selectin could be used for MSC separation.17–20 A microfluidic channel can achieve a large surface area-to-volume ratio and will be a good platform for effective cell-channel interaction and subsequent cell rolling behavior. Moreover, a


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microfluidic channel provides stable flow controllability and good microscopic visibility, and these features are very attractive to evaluate cell rolling behavior. In this study, we describe iPSC separation using a microfluidic channel with an immobilized monoclonal antibody (mAb) to stage-specific embryonic antigen 1 (SSEA-1; also called CD15 or Lewis X), which is a murine pluripotency membrane marker.8,11 To reduce non-specific protein adsorption and immobilize the mAbs covalently, the channel was coated with poly(2methacryloyloxyethyl

phosphorylcholine

[MPC]-co-n-butyl

methacrylate

[BMA]-co-p-

nitrophenyl oxycarbonyl poly[ethylene glycol] methacrylate [MEONP]) (PMBN).21,22 Then, mAb molecules were immobilized by an exchange reaction of amino groups of the mAbs and active ester groups of MEONP unit in the PMBN (Figure 1). Cell movement velocity in microfluidic channels with or without immobilized mAbs was investigated using a fluorescent microscopy by injecting two types of cells: iPSCs and NIH/3T3 fibroblasts (3T3s) as a control. By fractionating cells eluted from the channel, the proportions of iPSCs were measured using flow cytometer. We thereby evaluated whether the immobilized-antibody triggered iPSC rolling and iPSC was separated on the surface due to the cell rolling behavior.


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Figure 1. Synthesis of PMBN and immobilization of anti-SSEA-1 antibody.

EXPERIMENTAL SECTION

Materials. P-nitrophenylchloroformate and trimethylamine (TEA) were purchased from Sigma-Aldrich Co., LLC (St. Louis, MO). Poly(ethylene glycol) methacrylate (Blenmer PE-200) was obtained from NOF Co. (Tokyo, Japan). MPC was purchased from NOF, which was synthesized previously reported method22. BMA was purchased from Kishida Chemical Co., Ltd. (Osaka, Japan). Custom-made glass microfluidic channels were purchased from the Institute of Microchemical Technology Co., Ltd. (Kanagawa, Japan). Mouse iPSCs (iPS-MEF-Ng-178B-5) were purchased from the RIKEN BioResource Center (Ibaraki, Japan). NIH/3T3 cells (clone 5611) were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). Anti-human/mouse SSEA-1 mAbs with and without a phycoerythrin (PE)-label were purchased from eBioscience, Inc. (San Diego, CA).


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Synthesis of MEONP. MEONP monomer was synthesized based on the methods reported previously.21,23 Briefly, p-nitrophenylchloroformate (10.1 g) was dissolved in 100 mL chloroform and the solution was cooled at -30°C. Poly(ethylene glycol) methacrylate (14.2 mg) and TEA (6.05 g) dissolved in 50 mL chloroform were added using a dropping funnel for 1.0 h and the reaction mixture was stirred for 24 h at -30°C. After the reaction, above 70% of the solvent was evaporated and 150 mL diethyl ether was added to precipitate TEA hydrochloride. After filtration the solvent was evaporated and then added diethyl ether again. This process was repeated twice. After filtration, the filtrate solution was washed 3 times in a separating funnel with 50 mL hydrochloric acid (10 mmol/mL) to remove remaining TEA completely. The organic phase was dehydrated using anhydrous magnesium sulfate for 2 h. After filtration and evaporation, MEONP was obtained as a light yellow oily liquid. The structure of MEONP was confirmed by 1H-nuclear magnetic resonance (NMR) measurements (Gemini 2000/300; Varian, Inc., Palo Alto, CA) and Fourier transform infrared spectroscopy (FT/IR-6300; JASCO Corporation, Tokyo, Japan). Synthesis of PMBN. Conventional radical polymerization was performed to synthesize a copolymer composed of MPC, BMA, and MEONP using 2,2′-azobisisobutyronitrile (AIBN) as an initiator (Figure 1), as described previously.21,23 Briefly, 0.70 mol/L of mixed monomer solution in ethanol was prepared with the addition of 7.0 mmol/L AIBN, and the reaction was performed for 15 h at 60°C. After polymerization, the reaction mixture was precipitated using a mixture of chloroform and diethyl ether (20:80). The monomer unit mole fraction in the obtained PMBN was determined using 1H-NMR in ethanol-d6 (C/D/N Isotopes, QC, Canada). Molecular weight was examined using gel permeation chromatography (Shodex SB804-HQ; Showa Denko KK, Tokyo, Japan) by dissolving in an eluent (MeOH:pure water 70:30, including 10 mM LiBr).


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A poly(ethylene oxide) standard (TSKstandard poly(ethylene oxide); Tosoh Corporation, Tokyo, Japan) was used to calculate molecular weight. Two types of polymer with and without MEONP unit (PMBN and PMB, respectively) were synthesized (Table 1). The contact angles of PMBNand PMB-coated cover slips were measured using a captive bubble method (CA-X; Kyowa Interface Science, Saitama, Japan).

Table 1. Results of PMBN synthesis. Monomer composition [mol%] In feed

Mw (x104)

In composition

MPC

BMA

MEONP MPC

BMA

MEONP

PMBN

30

60

10

37

60

3.4

5.1

PMB

33

67

0

36

64

-

3.3

Antibody immobilization to microfluidic channels. The inner wall of a microfluidic channel was coated with PMBN or PMB. Filtered polymer solution in ethanol (0.20 wt%) was pipetted into a channel and incubated for 1.0 h at room temperature in the dark, before the solution was removed and the chip was dried under vacuum overnight. Subsequently, 10 µg/mL anti-SSEA-1 mAb solution in phosphate buffered saline (pH 8.0) was pipetted into the channel and allowed to react with the active ester groups on MEONP units for 24 h at room temperature (Figure 1). The chip was assembled with a chip holder and poly(ether ether ketone) (PEEK) capillary tubes (Figure 2A). Prior to the experiments, Hank’s balanced salt solution (HBSS; Sigma-Aldrich Co., LLC, Tokyo, Japan) was applied at a flow rate of 10 mL/h using a syringe pump (Model: 780120 J; KD Scientific, Inc., Holliston, MA) for 1.0 h so as to flush out unreacted antibody residues.


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Figure 2. (A) Glass based microchip and PEEK inlet and outlet tubes were assembled with a chip-holder. The width and height of the microfluidic channel was 340 and 170 µm, respectively. Scale bar, 10 mm. (B) Experimental procedure of cell injection into the microfluidic channel. Preparation of iPSCs. Mouse iPSCs, in which the green fluorescent protein (GFP) reporter gene is driven by the Nanog promoter, were cultured as described previously.24 The cells were seeded at a density of 5.7 × 103 cells/cm2 on mitomycin-C-treated SNL feeder layers and maintained in undifferentiating medium (Dulbecco’s modified Eagle’s medium [DMEM] high glucose; Gibco, Thermo Fisher Scientific, Inc., Waltham, MA) with 15% fetal bovine serum


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(FBS; Equitech-bio, Inc., Kerrville, TX), 0.1 mM non-essential amino acids solution (100×; Gibco, Thermo Fisher Scientific, Inc., Waltham, MA), 2 mM L-glutamine (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA), 0.1 mM 2-mercaptoethanol (Gibco, Thermo Fisher Scientific Inc., Waltham, MA), and 1,000 U/mL leukemia inhibitory factor (ESGRO; Merck Millipore Corporation, Darmstadt, Germany) at 37°C in a humidified atmosphere with 5% CO2. The medium was changed every day except day 1. After 4 days culture, the crude iPSCs were dissociated using a 0.25% trypsin-EDTA solution (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA), and suspended in HBSS containing 1% Pen-Strep (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA) at a density of 2.0 × 107 cells/mL. Nanog-GFPPos iPSCs were isolated using FACSAria IIIu (Becton, Dickinson and Company, Franklin Lakes, NJ). 3T3s were cultured at 37°C in a 5% CO2 atmosphere with DMEM supplemented with 10% FBS and 1% Pen-Strep. Preconfluent cells were detached using 0.05% trypsin-EDTA (Gibco, Thermo Fisher Scientific Inc., Waltham, MA) and dyed with fluorescent CellTracker BODIPY green (Molecular Probes, Thermo Fisher Scientific, Inc., Waltham, MA) prior to the experiments. The cells were suspended in HBSS containing 1% Pen-Strep before the experiments at a density of 2.0 × 107 cells/mL. Cell diameters were measured using a Z2 Coulter counter (Beckman Coulter, Inc., Hialeah, FL). Pluripotency assay of iPSCs by FACS. The undifferentiated states of crude iPSCs were measured using a Stemflow Human and Mouse Pluripotent Stem Cell Analysis Kit (Becton, Dickinson and Company, Franklin Lakes, NJ) in accordance with the kit’s instructions. Briefly, the cells were fixed with 4% paraformaldehyde and permeabilized with 1× Parm/Wash buffer. Each cell sample was stained with a PE-conjugated anti-SSEA-1 antibody (1:5) and PerCP-


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Cy5.5-conjugated anti-Oct3/4 antibody (1:5), and the expression of each marker was measured using FACSCalibur (Becton, Dickinson and Company, Franklin Lakes, NJ). Measurement of velocity and transit time of cell movement. Nanog-GFPPos iPSCs or fluorescent-dyed 3T3s were applied to a PMBN- or PMB-coated microfluidic channel, and cell movement velocity was measured. The experimental procedure is described in Figure 2B. Cell suspensions (from 1 to 2 µL of 2.0 × 107 cells/mL) were injected into the antibody-treated microfluidic channels. The injected cells were placed within 30 mm downstream of the confluence of the cell and eluent injection channels, and allowed to stand still without eluent flow for 1 min, in order to let the cells sediment to the bottom of the channel. Subsequently, 0.5 mL/h eluent flow (HBSS with 10% FBS and 1% Pen-Strep) was applied using a syringe pump. A 3-way cock was used to switch on/off the eluent flow. Cell movement at the observation point, located 100 mm downstream of the confluence, were captured using a high-speed CCD camera (EM-CCD digital camera; Hamamatsu Photonics K.K., Shizuoka, Japan) mounted on an inverted microscope (Eclipse TE2000; Nikon Instech Co., Ltd., Tokyo, Japan) and exposed to an emission light with a wavelength of 505 nm. Captured images were processed using ImageJ (http://imagej.nih.gov/ij/; National Institutes of Health, Bethesda, MD) to improve cell visibility by subtracting subsequent frames. Every captured cell was tracked manually and velocity of cell movement was measured using MTrackJ.25 We also recorded the frame number when each cell appeared, to measure transit time from the start position to the observation point. Fractionation of iPSCs using microfluidic channel. Crude cell suspensions, containing iPSCs, SNL feeder cells and partially differentiated cells, were injected into a PMBN-coated microfluidic channel with the same procedure as described in the previous section. The eluted


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cells were fractionated into collection tubes by switching the tubes every 30 s. This procedure was repeated 30 times with a 1-min interval between procedures. The fractionated cells were stained with a PE-labeled anti-SSEA-1 mAb (1:20), and the expression levels of SSEA-1 and Nanog in each aliquot (at an elution time of 0–90, 90–120, 120–150, 150–180, 180–210, 210– 240, 240–270, and 270-360 s) were measured using FACSCalibur. Statistical analysis. Significant differences of contact angle and movement velocity of cells were checked using Welch’s t-test. Cohen’s d26 was calculated in order to evaluate the quantitative effect of different channel surfaces on cell movement velocity. All statistical tests were performed with a 95% confidence interval (CI).

RESULTS AND DISCUSSION

Polymer preparation. The monomer unit composition and molecular weight for PMBN and PMB are shown in Table 1. The present synthetic results of PMBN were in good agreement with those in previous studies.21,23 No significant difference was observed between the bubble contact angle on PMBN- and PMB-coated glass surfaces (100.0 ± 5.9 and 106.0 ± 6.1, respectively), and it can be supposed that hydrophilic properties scarcely affect cell-substrate interactions. In our previous study,27 we approved phospholipid polymer as a CRC-coating substrate, which suppressed nonspecific cell adhesion to the channel surfaces. The active ester group on MEONP unit provides a covalent bond to the amino groups of antibodies. Consequently, PMBN was used for the microfluidic channels with immobilize-antibody (mAb+), and PMB was used for those without immobilize-antibody (mAb-) as a negative control. Phenotypic assays of cells. The Oct3/4 and SSEA-1 expressions of crude iPSCs are shown in Figure 3. Oct3/4 is a transcription factor associated with establishment and maintenance of cell


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pluripotency.28,29 SSEA-1 is a carbohydrate membrane antigen expressed at the early development stage of mouse cells, such as embryonic blastomeres11, embryonic stem cells, and iPSCs.8 The proportion of Oct3/4Pos cells was higher in the SSEA-1Pos group than in the SSEA1Neg group, indicating that it is possible to enrich pluripotent cells by selecting SSEA-1pos cells. Stadtfeld et al. reported that SSEA-1 is an enrichment marker for iPSCs by upregulating an early step of iPSC reprogramming.8 From these results, we chose SSEA-1 as a membrane marker for iPSCs. Additionally, 3T3s were negative for SSEA-1 and served as a negative control (Figure S1). The size of iPSCs and 3T3s was estimated using a Coulter Z2 particle counter and size analyzer (Figure 4). The diameter of 3T3s (15.2 ± 2.1 µm) was approximately 1.7 times larger than that of iPSCs (9.1 ± 1.1 µm).

Figure 3. The SSEA-1 and Oct3/4 expression of crude iPSCs measured by FACSCalibur. The proportion of each quadrant was 28% (SSEA-1Pos/Oct3/4Pos; S+/O+), 1.9% (S+/O-), 55% (S/O+), 16% (S-/O-).


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Figure 4. Size measurement of iPSCs and 3T3s using a Coulter Z2 particle counter and size analyzer. The diameter of 3T3s and iPSCs was 15.2±2.1 and 9.1±1.1 µm, respectively (mean±standard deviation). Velocity of cell movement in mAb-immobilized channels and SSEA-1 expression. We measured velocity of cell movement under constant flow conditions in the microfluidic channels with or without the immobilized-antibody against SSEA-1 (mAb+ or mAb-, respectively). Each cell movement was observed with a fluorescent microscopy (Video 1), and the movement velocity and transit time of each cell was measured (Figure 5). Cohen’s d of the difference between the 3T3-velocity in the mAb+ and mAb- channel was 0.31, which can be interpreted as that the immobilized-antibody had a small effect on the movement velocity of 3T3s.26 In contrast, the iPS-velocity in the mAb+ channel was significantly lower than in the mAb-, and Cohen’s d of the difference between velocities of cell movement in the mAb+ and mAb- channel was 2.45 in the iPSC group, which was much larger than in the 3T3 group. In accordance with


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the iPSC-velocity suppression, the transit time was increased dramatically. From this, we concluded that the microfluidic channel with immobilized anti-SSEA-1 antibody reduced the velocity of iPSCs and can be used for iPSC separation. Next, we performed a fractionation of crude cell suspension, which contained iPSCs with feeder cells and partially differentiated cells, using the mAb+ channel. The expression of SSEA1 and Nanog in fractionated cells was evaluated using a flow cytometer (Figure 6A). The proportion of SSEA-1Pos cells was increased from 1.6% in fractions 1–3 to 82% in fractions 10– 12 as a function of elution time (Figure 6B). The SSEA-1pos proportion of the crude cell suspension was 43%, indicating an enrichment of SSEA-1pos cells from the crude cell suspension. The movement velocity of the cells was inversely proportional to the transit time (Figure 5), and from this result, it can be assumed that the cells moved through the length of the channel at a constant velocity. It took an estimated 4.2 min for an iPSC, moving at 0.4 mm/s, to pass through the channel. This calculation is consistent with the result that the average expression of SSEA-1 was the highest in fraction 9 (equating to 4–4.5 min) (Figure 6C). The estimated medium flow rate distribution in the channel cross-section was calculated using the formula given by Cornish,30 under the assumption of a rectangular channel with a height of 170 µm and width of 340 µm (Figure S2). The average velocity of iPSCs in the mAb+ channel was 0.4 mm/s, and as fast as the average flow rate within 10 µm of the wall, implying that iPSCs stayed close to the channel wall due to the immobilized-antibodies against SSEA-1. From these results, we concluded that microfluidic channels with the immobilized-antibody specifically suppressed SSEA-1pos cell by producing the deterministic cell rolling, which can be used for label-free cell separation.


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Figure 5. Main panel, scatter plot of movement velocity and transit time of 3T3s or iPSCs in the mAb+ and mAb- channels. The velocity of cell movement was measured from microscopic images, and transit time was determined as the time from the loading region to the observation point. Each sample size is described in parentheses. Inset, Welch’s t-test was used to check the significance of movement velocity between 3T3s in mAb+ and mAb- (†p = 0.002, d = 0.31, 95% CI); between iPSCs in mAb+ and mAb- (*p = 0.000, d = 2.45, 95% CI); and between 3T3s and iPSCs in mAb+ (#p = 0.000, d = 1.36, 95% CI).


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Figure 6. (A) Flow cytometric analysis of iPSCs fractionated using the mAb+ channel. (B) The proportion of SSEA-1Pos cells in each fraction increased gradually as a function of elution time. The horizontal broken line corresponds to the proportion of SSEA-1Pos cells in pre-fractioned crude cell suspension. (C) The mean expression level of SSEA-1 in each fraction was calculated from geometric mean of PE fluorescence intensities using a flow cytometer. The horizontal broken line corresponds to the mean expression level of SSEA-1 of pre-fractioned cells. Effect of cell size on cell fractionation with microfluidic channel. Interestingly, there was also a large difference between the velocity of 3T3s and iPSCs in the mAb- channels. This result suggested that cell size can be another factor of cell separation of this system. According to the estimated flow rate distribution (Figure S2), the average velocity of 3T3s was 3.4 mm/s,


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indicating that the majority of 3T3s were located near the centerline of the mAb- channel because the flow rate at the channel centerline was 3.3 mm/s, while that of iPSCs was 2.1 mm/s, indicating that iPSCs were located approximately 50 µm below of the channel centerline. The measurement of cell size revealed that 3T3s were 1.7 times larger than iPSCs in diameter. It was reported that bigger particles migrate closer toward the channel centerline under developed laminar flow conditions,31 due to the “tubular pinch effect”,32 and this is consistent with the estimation of cell position described above. One possible reason of the cell-velocity difference is that big 3T3s were located closer to the center of the channel and experienced faster surrounding flow than small iPSCs in the equilibrium state. In other studies, it was also reported that the equilibrium position of a particle in a microfluidic channel varies as a function of its size and deformability in viscosity-dominated flow,33 and moreover, these effects have been investigated for cell separation techniques.34–36 It is noteworthy that the size effect must be carefully investigated, and especially in case where contaminant cell was smaller than iPSCs, the effect may decrease the efficiency of cell separation. In the present results, the movement velocity of SSEA-1Pos cells rolling on the mAb+ channel was outstandingly low compared with the cell movement velocity in the other groups, and sizeeffect will not interfere with the cell separation performance of this microfluidic system. To conclude, cell rolling behavior on microfluidic channels with immobilized-antibody will be a criterion for label-free iPSC separation, and CRCs constructed with microfluidic channel will be a promising platform for cell separation. Pluripotency of cell fractionated with microfluidic channel. The pluripotency of the separated cells was remained to be discussed, because, in Figure 6A, some of the cells in the fraction 10–12 showed little Nanog expression, which is a homeobox protein involved with stem


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cells and widely used as a pluripotency marker. Our result may suggest that some cells lost their pluripotency in this later fraction. Further experiments are needed to check pluripotency of the separated iPSCs, and to find better affinitive molecules with high selectivity for stem cells. Processing speed of present system. The processing speed of this system was limited to approximately 250 cells/min (1,500 cells per 6 min, as shown in Fig. S3), which was much lower than that of FACS or other cell sorting systems37. In clinical application, it was said that 107–108 cell are required to regenerate injured tissues38, and to achieve that quantity, it is necessary to increase the surface area of channel wall by parallel channel accumulation. Another possible application is single-cell cloning techniques. On-chip single-cell-based screening and cloning will be realized by this technique.

CONCLUSIONS

We performed label-free iPSCs separation using the PMBN-coated microfluidic channels with immobilized-antibody against SSEA-1. The velocities of iPSCs and 3T3s movement under a liquid flow condition in the channel were measured by microscopic observation. The mean velocity of iPSCs was decreased to 21% by the immobilized-antibody, whereas that of 3T3s did not change with the antibody immobilization. There was also a difference between the mean velocity of 3T3s and iPSCs (3.4 and 2.1 mm/sec, respectively) in the unmodified channel. These results suggested that the expression of membrane marker and cell size were determinate factor of cell movement in this system. By fractionating the heterogeneous cell suspension which was 43% SSEA-1pos, the lately eluted cell fraction containing 81% of SSEA-1pos iPSCs could be obtained. These results indicated that the microfluidic channel immobilized with anti-SSEA-1


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antibody selectively reduced the movement velocity of SSEA-1Pos cells by producing deterministic cell rolling, and this system can be used for label-free iPSC separation based on the SSEA-1 expression.


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ASSOCIATED CONTENT Supporting Information. The full results of FACS analysis (Figure S1), and the estimated flow rate distribution (Figure S2). (PDF) The observation results of cell movement (Video 1). (AVI)

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT

I would like to thank Dr. Ji-Hun Seo from the University of Tokyo for his advice for synthesizing the polymer. This work was supported by a Grant-in-Aid for Young Scientists (B) (No. 15K21696) from the Japan Society for the Promotion of Science, and by the Intramural Research Fund (26-6-22, 28-6-8) for Cardiovascular Diseases of National Cerebral and Cardiovascular Center.


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COT Graphic


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Label-Free Separation of Induced Pluripotent Stem Cells with Anti

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