A New Cell Separation Method Based on Antibody-Immobilized

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A new cell separation method based on antibody-immobilized nanoneedle arrays for the detection of intracellular markers Ryuzo Kawamura, Minami Miyazaki, Keita Shimizu, Yuta Matsumoto, Yaron R Silberberg, Ramachandra Rao Sathuluri, Masumi Iijima, Shun'ichi Kuroda, Futoshi Iwata, Takeshi Kobayashi, and Chikashi Nakamura Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b03918 • Publication Date (Web): 19 Oct 2017 Downloaded from http://pubs.acs.org on October 19, 2017

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A new cell separation method based on antibodyimmobilized nanoneedle arrays for the detection of intracellular markers Ryuzo Kawamura1†, Minami Miyazaki2, Keita Shimizu2, Yuta Matsumoto2, Yaron R. Silberberg1, Ramachandra Rao Sathuluri1, Masumi Iijima3, Shun’ichi Kuroda3, Futoshi Iwata4, Takeshi Kobayashi5 and Chikashi Nakamura1,2* 1

Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central5 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8565, Japan

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Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-26 Naka-cho, Koganei, Tokyo, 184-8588, Japan

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Department of Biomolecular Science and Reaction, The Institute of Scientific and Industrial Research (ISIR-Sanken), Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0046, Japan

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Department of Mechanical Engineering, Shizuoka University, 3-5-1 Johoku, Hamamatsu 4328561, Japan

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Research Center for Ubiquitous MEMS and Micro Engineering, AIST, 1-2-1, Namiki, Tsukuba, Ibaraki 305-8564, Japan

* Corresponding Author, Phone +81-29-861-2445, E-mail: [email protected]

ACS Paragon Plus Environment

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ABSTRACT

Focusing on intracellular targets, we propose a new cell separation technique based on a nanoneedle array (NNA) device, which allows simultaneous insertion of multiple needles into multiple cells. The device is designed to target and lift (‘fish’) individual cells from a mixed population of cells on a substrate using an antibody-functionalized NNA. The mechanics underlying this approach were validated by force analysis using an atomic force microscope. Accurate high-throughput separation was achieved using one-to-one contacts between the nanoneedles and the cells by preparing a single-cell array in which the positions of the cells were aligned with 10,000 nanoneedles in the NNA. Cell-type-specific separation was realized by controlling the adhesion force so that the cells could be detached in cell-type-independent manner. Separation of nestin-expressing neural stem cells (NSCs) derived from human induced pluripotent cells (hiPSC) was demonstrated using the proposed technology, and successful differentiation to neuronal cells was confirmed.

KEYWORDS: cell separation, intracellular marker, nanoneedle array, single-cell array, iPS cells, high-throughput


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Stem cell technologies, such as the production of embryonic stem cell (ESC), mesenchymal stem cell (MSC) and induced pluripotent stem cell (iPSC), have opened a new medicinal science field that encompasses regenerative medicine and drug discovery1-9. Efforts focused on the effective use of these pluripotent cells involve not only the control of differentiation and the fate of the cells, but also the separation of target cells from a heterogeneous population of differentiated cells. Safe cell transplantation, in which undesirable differentiated cells and undifferentiated tumorigenic cells are excluded, requires an accurate and versatile cell sorting technology that can deal with vast cell populations in a high-throughput manner. Fluorescently activated cell sorting (FACS) is the most powerful tool for this application presently available 10 and allows high-throughput cell sorting by detecting cell surface protein markers. However, it remains difficult to sort specific cells efficiently because the molecular target must be displayed on the cell surface, such as cluster of differentiation molecules (CD molecules). Attempts to use multiple makers for the purification of neuronal stem cells (NSCs) by FACS may overcome the limited specificity of surface markers, but clinical application remains difficult due to the lack of a method for removing antibodies bound to the cell surface 11,12

. Surface-displayed markers are widely used in FACS whereas intracellular markers have not

been targeted due to their relative inaccessibility. Intermediate filament (IF) is a category of cytoskeletal protein and consists of over 50 different proteins, of which some are markers for a given cell type 13. Vimentin and nestin are both IFs, and nestin is reported to be a good candidate marker of NSCs as it is temporally expressed during the neural differentiation process from pluripotent stem cells 14,15. The detection of nestin in differentiating cells could be a key step in realizing the effective purification of neural progenitor cells, thereby holding promise for safer cell transplantation therapies for neural defects such as spinal cord injury 16,17. We were therefore


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motivated to develop a cell separating method that detects nestin in living cells in a highthroughput manner. Approaches for accessing the intracellular environment using ultrathin probes inserted into cells have been reported as sensing tools and encompass electro-chemical sensing, optical sensing, and surface-enhanced Raman spectroscopy 18-20. We have developed a mechanical approach using an ultra-thin probe or ‘nanoneedle’ 21,22. Using an atomic force microscope (AFM), a nanoneedle can be effectively inserted into substrate-adhering cells 23. The nanoneedle is 200 nm wide and over 10 µm long, and can be inserted into living cells, as well as penetrating into the nucleus, but without affecting the doubling time of the cells or causing double-stranded DNA breaks 24,25. Functionalization of the nanoneedle with antibodies allows the detection of cytoskeletal proteins such as nestin by force spectroscopy AFM during removal of the inserted nanoneedle 24,26. Furthermore, the use of a hook-shaped nanoneedle allows the detachment and lifting (termed ‘fishing’) of substrate-adhering cells 27. Following these observations, we anticipated that cells could be separated using antibody-modified nanoneedles due to specific interactions between the nanoneedle and intracellular molecules. This approach would be compatible with intact living cells and would allow cell separation that needs neither transformation of the cells for fluorescent visualization of nestin expression, nor removal of fluorescently-labeled antibodies from the plasma membrane, as in the case of the FACS method, since the antibodies used in our method, for the detection of intracellular marker proteins, are covalently attached to the nanoneedle15. The high-throughput required for cell separation is very difficult to achieve using an AFM-based operation and thus we developed a nanoneedle array (NNA) technology 28 to manipulate a large number of cells in an arrayed form simultaneously, as depicted in Figure 1. Accurate separation using an NNA comprising of 10,000 nanoneedles


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required developing a single-cell array distributed as an adhesive spot pattern, thereby providing uniform one-to-one contacts between the nanoneedles and the cells. In this report, we demonstrate the nestin-targeted separation of NSCs in the process of differentiating from human induced pluripotent cells (hiPSCs) as a proof-of-concept of a new cell separating method utilizing intracellular markers. To this end, the ‘fishing force’ holding the cells to the nanoneedle through antigen-antibody interactions and the ‘adhesion strength’ keeping the cells adhered on the substrate were finely tuned based on mechanics revealed by AFM force measurements of one-to-one interactions between the nanoneedle and the cell. Vimentin and nestin were mechanically distinguished using positive- and negative-control cell lines. The targeting and removal (‘fishing’) of individual cells from a substrate requires that the ‘fishing force’ should exceed the cell–substrate adhesion force (Figure 1A). We first analysed the mechanics of this operation and optimized the conditions for preparing antibody-modified nanoneedles and the adhesion-controlled substrate. The fishing force was measured as previously reported but using the bio-nanocapsule anchoring agent ‘ZZ-BNC’, which consists of liposomes decorated with the IgG binding domain, thereby allowing multiple one-to-one bindings of IgG and the target molecule 24,29-31. Using an AFM system equipped with an antibody-immobilized nanoneedle, the force profile during nanoneedle insertion into and retraction from a live cell adhering to a dish was recorded (Figure 2A). The fishing force was determined by the peak force on the force–distance curve obtained during the needle retraction process from the adhering cell (Figure 2B). Although the mechanics underlying the detachment event is usually described by the work energy calculated from the force curve, in this paper the cell fishing event is described by the peak force due to the simplicity of this approximation and the reasonable correlation between the peak force and the detachment energy (See also Supporting Information, Figure S1).


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Two different IFs were selected as targets to demonstrate the versatility of this method. First, the mechanics of the fishing force targeting vimentin, which is immunohistologically well known but their function remains unclear, was investigated using the vimentin positive and negative human cell lines HeLa and MCF-7. The average fishing force was 12.1 ± 7.1 nN (n = 30), with a maximum value of over 30 nN, for HeLa using an anti-vimentin antibody-modified nanoneedle, whereas MCF-7 cells provided an average value of 5.7 ± 2.4 nN (n = 27) and a maximum value of less than 10 nN (Figure2C). A negative control nanoneedle was modified only with ZZ-BNC and provided average values of 3.6 ± 2.3 nN (n = 27) and 3.9 ± 2.4 nN (n = 30) for HeLa and MCF-7 cells, respectively. Since the force was always less than 10 nN, these results support the specificity of antibody-immobilized nanoneedles. The second model IF, nestin, was targeted using the nestin positive and negative mouse cell lines P19 and NIH3T3. Fishing force measurements using anti-nestin antibody-modified nanoneedles provided results similar to those obtained using vimentin: over 30 nN maximum for P19 and less than 10 nN for NIH3T3 (Figure 2D). The average forces were relatively small: 6.4 ± 7.7 nN (n = 39) and 2.9 ± 1.9 nN (n = 24) for P19 and NIH3T3, respectively. A negative control without antibody modification showed the same maximum force: less than 10 nN for both P19 and NIH3T3 cells, and the average forces were 2.5 ± 1.8 nN (n = 30) and 3.0 ± 2.3 nN (n = 22), respectively. These results demonstrated that antibody-immobilized nanoneedles have potential for ‘fishing’ and lifting target cells, if the adhesion force could be controlled to 10 nN. Next, we tried to control the adhesion force on cell arrays. Our aim was to fabricate an array of 10,000 nanoneedles to ‘fish’ through one-to-one contacts with live cells using antibody-antigen binding. We therefore prepared a detachable single-cell array using a previously reported method in which the density of biocompatible


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anchor for membrane (BAM) was tuned on a ‘cell-repulsive’ BSA coating 32,33. An adhesive spot array was prepared by micro-contact printing using a micro-pillar array (MPA). This MPA was an intermediate in the generation of an NNA (Figure 3A–C, Figure S2). A liquid thin film of BAM ink was formed by spin-coating and the ink was printed as a dot pattern onto a BSA-coated dish using the MPA on the microscope manipulator. The array of 3.9 µm diameter micro-pillars generated spots 4.7 ± 0.4 µm in diameter (Figure S3). BAM was reacted with BSA at room temperature overnight to provide cell-adhesive spots displaying oleyl groups on the BSA-coated substrate. To prevent cell-type-dependent adhesion through their specific surface-displayed proteins, cells cultured to adhere on normal cell culture dish were treated with Accumax for 10 min and plated on the spot-printed dish in medium supplemented with 10 mM EDTA but lacking FBS. Suspended cells were removed by rinsing with medium to provide the cell array. Up to 90% of the spots were occupied by cells (Figure 3D; see also Figure S4 for a magnified image) and the cells were well-dispersed to single state without contacting each other at the spots of 30 µm pitch. The cells in the array should be aligned with the nanoneedles in the NNA, and thus the accuracy of positioning the cells at the targeted locations was evaluated (Figure 3E). At the lowest BAM concentration (0.5 mM) over 10% of the cells were positioned inaccurately. Plotting the adhesion strength against the BAM concentration (Figure 3F) showed that the minimum adhesion strength was less than 20 nN at 2 mM BAM, and thus 2 mM BAM was deemed the lowest practical concentration for this application. Cell occupancy in the array at 2 mM BAM was approximately 40% due to the removal of unbound cells by hand pipetting; automation of this procedure may improve cell occupancy in future. Adhesion forces in the cell array were equalized between different cell types, although they differ by up to ten-fold on normal cell culture dish (Figure S5) 27. The adhesion force in the cell array resulting from


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physical tethering of the cell membrane with the BAM oleyl chain can be controlled independent of natural cell adhesion properties, consistent with previous report 33. Furthermore, it was found that decreasing the retraction speed of the nanoneedle from 10 to 0.1 µm/s increases the difference between the specific fishing force in the target-positive and the nonspecific interaction force in the target-negative cells (Figure S6A) because nonspecific interactions have a shorter bond lifetime. However, retracting the nanoneedle at 0.1 µm/s is time consuming (Figure S6B) and thus we used a retraction rate of 1 µm/s in the following experiments. After verifying the mechanics underlying the cell fishing process and the preparation of cell arrays, we next demonstrated proof-of-concept cell separating using model cell lines. An NNA was fabricated using a top-down MEMS technique, as described previously (Figure S2) 28. Each nanoneedle was less than 200 nm in diameter and more than 20 µm long. The approach of the NNA to the substrate is precisely controlled using a piezo motor-driven manipulating system. This allows accurate positioning of one-to-one contact of the cells and needles at all points of the array, as previously reported (Figure S7) 28. The NNAs were modified with antibody in the same manner as used for the single nanoneedles. Anti-vimentin antibodies were immobilized on the NNAs and HeLa cells were separated from a mixture of HeLa and MCF-7 cells (Figure 4A). The separated target cells were deposited into a collecting plate (Figure 4B) coated with a strong adhesive. The expression of vimentin and its fibrous structure was retained in HeLa cells on the arrayed form (Figure 4C). Analysis of the vimentin expression level by immunostaining following separation revealed that the separated cells expressed approximately 1.5 time more vimentin compared to cells remaining on the array (Figure 4D–F). Although the cell size seemed to affect differences in needle penetration depths, which may affect the fishing forces, there was no significant difference in size distribution of the cells between remained cells and collected


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cells after separation (Figure 4G). Thus, the target expression level seems to be the dominant factor in the separating criteria. The resulting efficiency of cell separation was calculated using the following formula: separationt [%] = (NCollected × 100 / (NCollected + NRemained), where NCollected is the number of cells on the collecting plate and NRemained is the number of cells remaining on the cell array. We found that 42% of the vimentin-positive HeLa cells were successfully separated and 7% of the negative control MCF-7 cells were collected despite the lack of specific interaction (Table 1); the percentage of collected target-positive cells was termed as ‘positive cell separation’ and that of collected negative control cells as ‘negative cell separation’. When anti-nestin antibody-modified NNA was used as a negative control, 4% of both HeLa and MCF7 cells were collected. The application of anti-nestin antibody-modified NNA to P19 and NIH3T3 cells provided 26% positive separation for P19 and 7% negative separation for NIH3T3 (Table 1), further confirming target-specific separation. The rather low ratio of positive separation to negative separation of nestin-positive cells can be attributed to the lower average fishing force (6.4 nN) compared to that of vimentin (12.1 nN). When the collected cells were supplemented with gelatin as an extracellular matrix and cultured on the same plate, they proliferated and showed elongated shapes, which is comparable to a normal culture (Figure S8). Our results demonstrate that an antibody-modified NNA can separate cells by their specific IF expression. Following our successful separation of HeLa and P19 cells, we attempted to separate nestin-expressing NSCs that had differentiated from hiPSCs. Using an established method, hiPSCs were differentiated to NSCs using serum-free PSC Neural Induction Medium 34 and the nestin-positive population was sorted using the nestin-targeted array system. Approximately 40% of the NSCs were successfully sorted to the strongly adhesive collecting plate (a glass plate


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functionalized with amino groups and densely coated with BAM) whereas less than 10% of the non-induced (undifferentiated) hiPSCs were collected. The correlation between the percentage of sorted cells and nestin expression both increased during the differentiation of NSCs from passage 0 to 4 (Figure 5A). Cell viability during this cell separation process was determined by staining the sorted cells with Calcein-AM: 97.5% of the sorted NSCs were alive, compared to 96.5% of normally cultured NSCs (Figure S9). These results further demonstrate that the nanoneedles used in this array technology do not harm cells, consistent with previous reports 21,22,24,35,36. To verify the ability of sorted nestin-positive NSCs to differentiate, the cells were further cultured on a laminin-coated substrate and subjected to neural differentiation. Differentiation was induced using Neurobasal medium supplemented with B-27, Glutamax-I, and penicillin/streptomycin, then the cells were observed after 14 days. The separated cells successfully differentiated into neurons that contained neurites and expressed neurofilaments (Figure 5B), comparable to cells that were differentiated under normal culture conditions without separation (Figure 5C). In contrast, non-induced, undifferentiated NSCs in medium without B-27 did not express neurofilaments (Figure 5D). IFs in cells with neurites were converted from nestin to neurofilaments after separation (Figure 5B), suggesting that these cells differentiated comparable to cells in normal culture (Figure 5C). When the immunostained images were quantitatively analyzed, the amount of neurofilament per cell area was found to be 35% (ncell =29) for the separated and differentiation-induced NSCs, while the normally-cultured cells resulted in 50% (ncell =13) with differentiation induction and 1% (ncell =57) without induction. Amount of neurofilament was evaluated as the area filled with bright pixels over a threshold. As reported by Song et al., successful neural differentiation from hiPS was determined by a neurofilamentpositive cell ratio of 43.7±12.3%, thus the separated cells using our NNA device would have


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enough potential for differentiation.37 Taken together, our results demonstrate that the proposed cell separating process using an anti-nestin targeted NNA sorts cells safely and correctly, and that the sorted cells retain the ability to differentiate. When the cells were cultured up to 13 days for further differentiation, the sorted cells appear to differentiate into neuronal cells more effectively, and with fewer round cells without neurites, compared to before separation or cells grown in normal culture (Figure S10). The results presented above demonstrated the principle of mechanical cell separation by the detection of intracellular IF markers. Since the antibodies are uniquely immobilized on the probe, there is no need to modify the cells with antibodies and wash them after separation, in contrast to FACS. Although separating efficiency must be improved to make this technology practical, our present results demonstrate the potential of this method for separating cells according to their intracellular target expression level and the fiber structure (Figure 4C–E). However, several major problems must be overcome. First, technical issues limiting separation efficiency and accuracy should be addressed, probably through optimized automated processing. The binding performance of the antibody-functionalized nanoneedles to the target cells must be improved while suppressing non-specific interactions to non-target proteins. Compared to the chemical crosslinkers used as IgG anchoring agents in our previous report, ZZ-BNC clearly increased the fishing force, minimizing non-specific interactions during needle insertion 24,26. Further improvement of the antibody-immobilizing method may increase the performance of our cell fishing approach. Second, the versatility of this method must be proven. We already demonstrated the examples of fishing force detection against actin and microtubule in the previous papers 24,26,38. For cell separation, molecules which bind to these cell body structures could be also targeted. According to the literature, cytoskeletal binding proteins constitute 3% of


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all proteins and structural cytoskeleton proteins constitute 8% of all proteins 39. Since the proteins located on the plasma membrane are estimated to constitute 8% of all proteins, antibodyfunctionalized nanoneedles can expand the target proteins for separation more than twice. Third, the applicability of this approach to human iPSCs is important. Dissociation of cell-cell interactions which is necessary to obtain single-cell array is known to be critical for human iPSCs, in contrast to mouse iPSCs. However, it was recently reported that Y-27632, a Rhoassociated coiled-coil forming kinase-inhibitor (ROCK-inhibitor), suppresses cell death by dissociation to single cells. This improvement in saving cells without cell-cell interaction may aid the application of our proposed method to human iPSCs. The ability to target nestin would be beneficial not only for regenerative medicine, but also for the diagnosis of cancer and the discovery of cancer therapies, since nestin is reported to be a candidate indicator of malignancy 40,41

. In an additional attempt to expand the range of target molecules to keratin, we are already

finding the potential versatility of this method (data not shown). We have demonstrated the application of nanoneedle technology to an array system with improved control of both the fishing force and cell adhesion, as well as mechanical cell separation by binding to two intracellular marker proteins. The cell separation efficiency of NSCs differentiated from hiPSC was 41%, and subsequent cultivation of the sorted cells resulted in effective differentiation to neuronal cells. These findings suggest the future applicability of the proposed technique to cell transplantation therapy following technical improvements and safety trials. Our approach is unique to conventional methods such as FACS and thus may be beneficial for sorting cells that are difficult to identify using only surface antigens. We will expand the range of target proteins and cells in the near future and improve nanoneedle functionalization.


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These advances may lead to the establishment of a new cell sorting technology applicable to cell transplantation therapy and the development of other new cell analysis technologies.


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Figure 1 | Schematics showing the principle of cell separation by intracellular makers using a nanoneedle array. (A) Mechanics of cell fishing by an antibody-immobilized nanoneedle. Adequate modification of the nanoneedles with antibodies and binding of the antibodies to the intracellular markers via antigen–antibody interaction allows the target cell to be mechanically fished from detachable cell adhesive spots. To fish nestin-positive cells, for example, the fishing force should exceed the cell adhesion force to the substrate. (B) Combination of a nanoneedle array and a single cell array allows simultaneous one-to-one contact for nanoneedle insertion into


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the cytosol of each cell. Preparation of the cell-adhering spot array with controlled adhesion in a cell-type-independent manner is critical for cell separation. (C) SEM images of a fabricated nanoneedle array with a 30 µm pitch. Scale bar is 20 µm.


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Figure 2 | Force measurement of single cell fishing with antibody-modified nanoneedles. (A) Setup for measuring the fishing force against an adhering cell using an antibody-modified nanoneedle attached to an AFM. (B) The fishing force is defined as the peak force in the retraction process on the force-distance curve as the cells remain attached to the substrate. The mechanics to fish the cells with an antibody-immobilized nanoneedle is evaluated by the peak force: this allows instant analysis, and gives information about the binding that is comparable to analysis by binding energy (see also Figure S1). Peak force was measured by retracting the nanoneedle at 1 µm/s to enhance the difference of the fishing force that is the total unbinding forces according to expression of the target protein (Figure S6). (C) The nanoneedle was modified with anti-vimentin antibody. For the negative control, the nanoneedle was modified with ZZ-BNC but not with antibody. Forces were measured for HeLa and MCF-7 cells; ‘inset’ shows the positive and negative vimentin expression of HeLa and MCF-7 cells, respectively. The horizontal line within each box indicates the median, the boundaries of each box indicate the 25th- and 75th-percentile, and the whiskers indicate the highest and lowest values obtained in the


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results. The average fishing force for HeLa cells was 12.1 ± 7.1 nN (average ± standard deviation) and 5.7 ± 2.4 nN for MCF-7 cells using antibody-modified nanoneedles. (D) Antinestin antibody was used for P19 and NIH3T3 cells. The average fishing force for P19 cells was 6.4 ± 7.7 nN and 2.7 ± 2.0 nN for NIH3T3 cells using antibody-modified nanoneedles. ‘inset’ shows the positive and negative nestin expression of P19 and NIH3T3 cells, respectively.


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Figure 3 | Preparation of a single-cell array by microcontact printing using a micropillar array. (A) SEM images of a fabricated micropillar array. The pillar array pitch is 30 µm. Scale bar is 20 µm. (B) Schematic showing substrate preparation for the single-cell array by microcontact printing of BAM onto a BSA-coated dish. BAM solution (0.5-10 mM BAM in glycerol:2-propanol:DMSO = 0.5:7:2.5) was printed as a dot pattern using a micropillar array. (C) A non-adhesive coating of BSA on the substrate was partially modified with BAM. This provided alkyl chains which can insert into the cell membrane in a cell-type-independent manner for controlled cell adhesion. (D) Overview of the cell array prepared with P19 cells. (E) Effect of the printing BAM concentration on accurate positioning of the cells in the targeted spot. The percentage of cells misplaced more than 5 µm from the target position was counted. (F) Cell


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adhesion force on the adhesive spots in the cell array was evaluated; the cell fishing assay used an AFM equipped with hook-shaped probes. The detachment force measured was plotted as adhesion force against BAM concentration. The percentage of cell occupation on the array spot at each BAM concentration was plotted along the right vertical axis.


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Figure 4 | Visual example of the cell separating process using the antibody-immobilized nanoneedle array. (A,B) Vimentin-targeted cell separation was performed using a cell array of HeLa and MCF-7 cells. (A) Immunostained cell array before separation. HeLa cells are visualized by the vimentin expressed and immunostained (green) while MCF-7 cells are visualized by Keratin expression and appear red. (B) The collected cells, deposited from the nanoneedle array onto a strong adhesion plate. This batch is different from that shown in (A). (C) Magnified image of vimentin in HeLa cells in the array. The fibrous structure seen in normal cultured cells is retained. (D,E) Vimentin expression levels of HeLa cells remained on the cell


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array plate (D) and those collected onto the nanoneedles (E); the x-y positions of these two images are not identical. (F) Fluorescence intensity of immunostained vimentin in each cell was quantitatively analyzed and plotted as histogram. Difference between Remained and Collected is significant as P < 0.04. (G) Distribution of the cell diameters of Remained and Collected HeLa cells. There is no significant difference in the diameters as P=0.58.


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Figure 5 | Neural stem cell separation using an anti-nestin antibody-immobilized nanoneedle array. (A) Percentage of sorted cells and nestin expression levels in hiPSCs and NSCs at passage 0 and 4 (P0 and P4, respectively) in PSC Neural Induction medium supplemented with Y-27632. (B–D) Cellular images obtained by phase contrast and immune staining with DAPI (blue), anti-Nestin antibodies (green), and anti-Neurofilament antibodies (red) after 14 days’ culture without or with induction of neural differentiation from hiPSCs. (B) The NSCs sorted onto the collecting plate using the nestin-targeted NNA were further cultured in medium containing B-27 supplement to induce differentiation into neural cells. (C) Normally cultured NSCs induced to differentiate in medium containing B-27, but without separation. (D) Normal culture of NSCs at passage 7 after culture in PSC Neural Induction Medium for differentiation into NSCs from hiPSCs, but without induction of further differentiation into neural cells with B-27.


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Table 1 | Efficiency of cell separation using the antibody-immobilized nanoneedle array.

Antibody’s target IF

Cell types Positive / negative

Positive† separation [%]

Negative† separation [%]

Positive/ Negative

Vimentin

HeLa / MCF-7

42.2±7.4

7.2±0.8

5.9

Nestin

P19 / NIH3T3

26.4±13.2

7.1±5.3

5.1

Nestin

- / HeLa, MCF-7

-

3.3±1.2, 3.2±2.1

-

†

Positive separation [%] is the value for the target IF-positive cells and Negative separation [%] is the value for IF-negative cells.


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ASSOCIATED CONTENT Supporting Information. Detailed description of the experimental methods and additional data are provided. This material is available free of charge via the Internet at http://pubs.acs.org. Following contents are included in a PDF file (NNA-Ab_SI.pdf). 1. Culture of HeLa, MCF-7, P19 and NIH3T3 cells 2. Nanoneedle fabrication from an AFM probe 3. Force analysis of cell fishing and adhesion with AFM 4. Preparation of nanoneedle array (NNA) and its manipulation for cell separation 5. Preparation of cell array and cell collecting plates 6. Culture of hiPSCs and induction of differentiation into NSCs 7. Immunostaining of the cells and imaging Figure S1–S10

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] Present Address †Department of Chemistry, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama, 3388570, Japan.


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Author Contributions C.N. conceived the method and organized the research; R.K., M.M., K.S., Y.M. and Y.S. performed the force measurements, cell separation, differentiation induction, and immunostaining experiments, and analyzed the data. R.S. optimized the protocols for fabricating the micropillar array and nanoneedle array. M.I. and S.K. designed and prepared ZZ-BNC. F.I. developed the array manipulator. T.K. designed the protocol to fabricate the nanoneedle array. R.K. and C.N. interpreted the data and wrote the manuscript. All the authors discussed the results and reviewed the manuscript.

Funding Sources This research was supported by the Japan Society for the Promotion of Science (JSPS) through the “Funding Program for Next Generation World-Leading Researchers (NEXT Program)”, initiated by the Council for Science and Technology Policy (CSTP).

Notes The authors declare no competing financial interests.


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for TOC only


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A New Cell Separation Method Based on Antibody-Immobilized

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