Controlled Cell Adhesion Using a Biocompatible Anchor for

May 3, 2013 - the desired proteins. By conjugating BAM with bovine serum albumin (BSA) absorbed on a dish for cell culture, both. Received: April 2, 2...
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Controlled Cell Adhesion Using a Biocompatible Anchor for Membrane-Conjugated Bovine Serum Albumin/Bovine Serum Albumin Mixed Layer Ryuzo Kawamura,† Mari Mishima,‡ Seunghwan Ryu,‡ Yu Arai,‡ Motomu Okose,‡ Yaron R. Silberberg,† Sathuluri Ramachandra Rao,† and Chikashi Nakamura*,†,‡ †

Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central4 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan ‡ Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-26 Naka-cho, Koganei, Tokyo 184-8588, Japan S Supporting Information *

ABSTRACT: We report here a method for controlling cell adhesion, allowing simple yet accurate cell detachment from the substrate, which is required for the establishment of new cytometry-based cell processing and analyzing methods. A biocompatible anchor for membrane (BAM) was conjugated with bovine serum albumin (BSA) to produce a cell-anchoring agent (BAM−BSA). By coating polystyrene substrates with a mixture of BAM−BSA and BSA, controlled suppression of the substrate’s adhesive properties was achieved. Hook-shaped nanoneedles were used to pick up cells from the substrate, while recording the cell−substrate adhesion force, using an atomic force microscope (AFM). Due to the lipid bilayer targeting property of BAM, the coated surface showed constant adhesion forces for various cell lines, and controlling the BAM− BSA/BSA ratio enabled tuning of the adhesion force, ranging from several tens of nano-Newtons down to several nano-Newtons. Optimized tuning of the adhesion force also enabled the detachment of cells from BAM−BSA/BSA-coated dishes, using a shear flow. Moreover, the method was shown to be noncell type specific and similar results were observed using four different cell types, including nonadherent cells. The attenuation of cell adhesion was also used to enable the collection of single cells by capillary aspiration. Thus, this versatile and relatively simple method can be used to control the adhesion of various cell types to substrates.

1. INTRODUCTION Control of cell adhesion in vitro is a key factor in the development of cell analyzing and processing technologies. Controlled adhesion to tether suspended cells in microfluidic devices may enable better image capture and lead to improved analysis, in comparison with flow cytometry analysis. Indeed, the detection of circulating tumor cells that are very rare or the sorting of differentiating stem cells without simple criteria remain difficult tasks using flow cytometry.1−5 On the other hand, tunable adhesion, which allows the controlled release of cells, could be beneficial for widening the applications of imaging cytometry. This would enable the precise analysis of adhering cells; however, it is not designed to enable the collection of cells following analysis.2,6 Establishment of a new method that will satisfy both these requirements by means of controlled, transient cell adhesion would be a key step forward in the development of a new technology to bridge the gap between the fields of flow cytometry and image cytometry. Such a fundamental technique would also contribute to the development of micrototal analyzing systems (μTAS).7−9 © XXXX American Chemical Society

Various methods for controlling cell adhesion have previously been developed, such as the use of Fibronectin (FN) and the RGD peptide-binding domain, collagen, poly-Llysine (PLL), hyaluronic acid (HA), and others.10−13 However, in these methods, the goal is usually to increase cell adhesion using substrates that are not designed to release cells. To realize a method for controlling cell adhesion for a wide variety of cell types, we focused on the membrane anchoring method reported by Nagamune et al.14 In this method, a material called a Biocompatible Anchor for Membrane (BAM) is used. This compound has three domains: a hydrophobic oleyl group for anchoring to the lipid bilayer of the cell, a hydrophilic polyethylene glycol (PEG) linker, and a functional group of Nhydroxysuccinimidylester (NHS), allowing covalent bonding to the desired proteins. By conjugating BAM with bovine serum albumin (BSA) absorbed on a dish for cell culture, both Received: April 2, 2013 Revised: May 2, 2013

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centrifuge. Harvested cells were resuspended in the medium and seeded onto culture dishes. 2.4. Subculture and Force Measurements. Cells cultured for 1−2 days in normal dishes were treated with 0.1% trypsin and 0.05% EDTA in PBS at 37 °C for 30 min in a 5% CO2 incubator, after rinsing in PBS. Equal volumes of trypsin inhibitor (R-007-100, Life Technologies) were added to the cell suspensions followed by centrifugation at 1000 rpm for 5 min. Cells were suspended in the medium without FBS but with 10 mM EDTA and seeded onto the BAM−BSA/BSA-coated dishes. Cells were then allowed to settle at 37 °C for 15 min in a CO2-controlled incubator prior to the experiment. 2.5. Preparation of Hook Needles. Hook-shaped nanoneedles (“hook needles”) were fabricated by etching the pyramidal tips of standard AFM silicon cantilevers (ATEC-Cont, Nanosensors, Switzerland) into flat arrowhead shapes with the use of a focused ion beam (FIB; SMI9200, SII). The width and height of the arrowheads were 1.6 and 2 μm, respectively, and the needles were approximately 8 μm in length and 300 nm in thickness. Spring constants (k = 0.4 ± 0.2 N/m) were determined using the thermal fluctuation method prior to each experiment.15 The nanoneedles were cleaned by immersing them in a sulfuric peroxide mixture (SPM, H2SO4:H2O2 = 4:1) at 55 °C for 30 min and then rinsing them twice in ultrapure water. 2.6. Adhesion Force Measurement Using Hook Needles with the AFM. AFM measurements were made on cells on BAM−BSA/ BSA-coated dishes after incubation at 37 °C for 15 min. The hook needle was operated by an AFM system (Nanowizard II, JPK Instruments) equipped with a CellHesion module, which enables the probe to have a vertical travel distance of up to 100 μm. Culture dishes were placed on a stage attached to an optical microscope (Axio Observer .A1, Zeiss), and force measurements were done within 1 h after starting the measurement at room temperature. The set point was set to a maximum force of 15 nN, and the needle was driven at a velocity of 5 μm s−1, during both the approach and retract processes. 2.7. Fluorescence Microscopy and Confocal Laser Scanning Microscopy. NIH 3T3 cells were plated on BAM−BSA/BSA-coated glass-bottomed culture dishes at 0.2−1 × 105 cells per 35 mm dish (3910−035, IWAKI), 1 day prior to Lipofectamine transfection. A mixture of 4 μg of plasmid DNA that codes for a red fluorescence protein (pPM-mKeimaRed, AM-V0253, Medical & Biological Laboratories) and 5 μL of Lipofectamine 2000 (11668, Life Technologies) were added to the 2 mL culture medium and incubated for 4 h. The medium was then replaced with fresh DMEM and incubated for 24 h in a CO2 incubator. To visualize hook-needle insertion to the adhering cell, the silicon surface was modified with a green fluorescent protein (GFP). A detailed protocol is given as Method S1 of the Supporting Information. Fluorescence images of the GFP-immobilized nanoneedle insertion into single cells transfected with pPM-mKeimaRed were acquired using a confocal laser-scanning microscope (CLSM; FV-300/IX71; Olympus, Tokyo, Japan) equipped with a CCD camera. Stacked CLSM images at 0.5 μm intervals were acquired, starting from 1−2 μm beneath the bottom of the cell to a vertical height of around 15−20 μm.

adherent and nonadherent cells were successfully immobilized. Here, we modify this method using a mixture of BAMconjugated BSA (BAM−BSA) and BSA for coating the dish, with the aim to tune the detachable adhesion of the cells to the substrate by simply changing the ratio of the BAM−BSA/BSA mixture (Figure 1).

Figure 1. The concept of cell adhesion control using a BAM−BSA/ BSA mixed layer.

2. MATERIALS AND METHODS 2.1. BAM−BSA Preparation. 1.05 mg mL−1 of BSA in 0.1 M bicarbonate (pH 8.5 by NaOH) was mixed with a 1/20 volume of 1.8 mM NHS-functionalized BAM (MW 2465; OE-020CS, NOF) solubilized in dimethyl sulfoxide (DMSO) and reacted at 25 °C for 1 h; the molar ratio of the final concentration was BSA:BAM = 1:6. After BAM−BSA was dialyzed in PBS (pH 7.4) without calcium to remove unreacted BAM, it was mixed with BSA in various ratios, and the total BSA concentration was diluted to 3 μM; as an example, a mixture containing a molar ratio of 2.5% of the obtained BAM−BSA and 97.5% of unmodified BSA was labeled “2.5% BAM−BSA”. The diluted mixture was prepared just prior to coating the dish. The labeling ratio of BAM to BSA was determined to be 4 ± 1 by molecular weight analysis using MALDI/TOF-MS (see Figure S1 of the Supporting Information). 2.2. Dish Coating with BAM−BSA/BSA Mixture and Cell Seeding. A polystyrene dish for cell culture (well size 35 mm in diameter) was placed with 2 mL of a BAM−BSA/BSA mixture diluted to 3 μM of the total BSA concentration and incubated at 37 °C for 3 h. After rinsing the dish 3 times with PBS using a 50 rpm rotary shaker for 5 min, 2 mL of each medium without FBS and with 10 mM EDTA was added and kept at 37 °C in a CO2 incubator until cell seeding. Since nontreated polystyrene substrates were not successfully coated with BAM−BSA/BSA, we always used dishes with corona-discharge treatment (3000-035, IWAKI, Japan). 2.3. Cell Culture. Mouse fibroblast National Institutes of Health (NIH) 3T3 (IFO50019, Health Science Research Resources Bank) and the human cervix carcinoma cell HeLa (RCB0007, Riken) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; D5546 Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS; 10437, Life Technologies), 2 mM GlutaMAX (050-061, Life Technologies), 10 mg mL−1 gentamicin, and 0.25 mg mL−1 amphotericin B (50-0640, Life Technologies). Mouse embryonic carcinoma P19 cells (EC95102107-F0, DS Pharma) were maintained in α-MEM (M4526, Sigma-Aldrich) supplemented with 10% FBS, 2 mM GlutaMAX, 10 mg mL−1 gentamicin, and 0.25 mg mL−1 amphotericin B. A cortical thymocyte cell line, JM (RCB0537, Riken), was maintained in RPMI1640 (R8758, Sigma-Aldrich) supplemented with 10% FBS, 2 mM GlutaMAX, and GA. One day prior to the experiment, the cells were treated with 0.25% trypsin-1 mM EDTA (25200-056, Life Technologies) and pelleted by

3. RESULTS AND DISCUSSION 3.1. Cell-Substrate Adhesion Measurements Using Hook Needles. BAM−BSA/BSA-coated dishes were prepared by adsorbing BAM−BSA/BSA mixtures with various ratios, while keeping the total BSA concentration at 3 μM. Polystyrene dishes were treated with either 2.5%, 5%, 10%, or 100% BAM− BSA. The distribution of the BAM−BSA appeared to be homogeneous and allowed for uniform cell adhesion, as no aggregation of BAM−BSA comparable to the cell size was observed when liposome containing fluorescein was immobilized on the substrates (Figure S2 of the Supporting Information).14 As shown in Figure S1 of the Supporting Information, four BAM molecules are immobilized on a BSA molecule. If the BAMs are equally distributed on the BSA B

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by mechanically detaching cells using an atomic force microscope (AFM) (Figure 2A). Previously, we reported that an AFM probe which had been sharpened to an ultrathin rod shape of 200−300 nm in diameter (i.e., a nanoneedle) could be inserted into live cells.16,17 This method is minimally invasive, as it was reported that the doubling time of the cells was not affected by repeated nanoneedle insertions.17 On the basis of this technique, we used a hook needle for picking up cells from the substrate while measuring their adhesion force to the substrate. The hook needle was fabricated by an FIB etching process (Figure 2B). Figure 2C shows an example of a force−distance curve obtained using the hook needle. The needle was moved toward the cells from 100 μm above and retracted to the original height after a dwell time of 2 s. In the approach curve, the force starts to increase as the needle displaces the plasma membrane, followed by a sudden steep drop in force, indicating penetration through the membrane. This penetration of the hook needle was confirmed by visualization with a CLSM, using a GFPmodified nanoneedle and cells expressing a cytosolic red fluorescent protein (KeimaRed) (Figure 2C, right image). During retraction of the nanoneedle, peaks were observed when the cells were detached from the substrate. Detachment of the cells was confirmed by phase-contrast microscopy. The minimum of the retraction curve peak was termed the “adhesion force” (Figure 2C). Adhesion forces ranged from several nano-Newtons and up to over 60 nN for both NIH 3T3 and P19cells (Figure 2D). Although the adhesion forces of NIH 3T3 cells were more than twice those of P19 cells in normal culture conditions on polystyrene dishes, their adhesion forces on BAM−BSA/BSA-coated dishes were almost equally controlled over a wide range of the BAM−BSA/BSA ratio; the same control of adhesion force could also be performed on glass bottom dishes (data not shown). The adhesion forces were almost constant with the time course during the 1 h of measurement, indicating that cell anchoring by BAM molecules reached a plateau within 15 min of the incubation before the measurement. Since the homogeneity of the BAM−BSA/BSA mixture layer had been confirmed (Figure S2 of the Supporting Information) and as the adhesion force is almost proportional to the BAM−BSA/BSA ratio (Figure 2D), the difference in adhesion force can be attributed to the difference in the density of surface-displayed BAM molecules. On the basis of this measurement, the performance of this technique for adhesion control was further investigated. As an AFM apparatus is not always readily accessible, a second evaluation method was applied, utilizing a shear flow of the medium. 3.2. Cell Detachment by Shear Flow. As controllable detachment and collection of the cells from the substrate is desirable for cell analysis and processing in microfluidics, we attempted to collect NIH 3T3 cells from the coated dish by the use of a microcapillary aspiration technique. As shown in Figure 3, cells were successfully collected using the capillary from the mildly adhesive 10% BAM−BSA dish, while neighboring cells were left undisturbed. This kind of cell collection by aspiration did not occur in the case of the 100% BAM−BSA-coated dish, even when the capillary was positioned in close proximity to the cell. Thus, adhesion control using BAM−BSA/BSA-mixed layer was found to be applicable for single cell control. Following this successful attempt of single cell collection, we next tried to detach cells by shaking the dishes to apply a shear flow of the medium. Four different cell lines, NIH 3T3, P19, HeLa, and JM (Figure 3A), were allowed to settle onto 10% or 100% BAM−

molecule, at least one BAM molecule can be accessible to the cell surface. With the assumption that the size of BAM−BSA is around 5 nm, the number of exposed BAMs on most tightly packed BAM−BSA layers is calculated to be around 5 × 104 molecules per square micrometer. To investigate the adhesion control of the mixture layer, two cell types were tested: fibroblast NIH 3T3 cells and mouse embryonic carcinoma P19 cells. After treatment with trypsin−EDTA, the cells were seeded on to each BAM−BSA/BSA-coated dish with serumfree medium supplemented with 10 mM EDTA; the latter was added to suppress the adhesion of proteins, such as cadherin, during the AFM measurements, which took up to 1 h. The ratios of settled/nonattached cells following incubation appeared to be correlated with the BAM−BSA/BSA ratios (Figure 2D, inset). On the 2.5% and 5% BAM−BSA dishes,

Figure 2. Measurement of the adhesion force of cell to a BAM−BSA/ BSA-immobilized surface. (A) Schematic illustration of cell fishing with the hook needle. (B) Hook needle fabricated by focused ionbeam etching. (C) Example of a force curve obtained during needle approach and retraction (inset picture); the needle surface was modified with GFP and the cell expressed KeimaRed by the introduction of plasmid DNA pPM-mKeimaRed. (D) Adhesion forces of NIH 3T3 (black) and P19 (red) cells for various BAM−BSA/BSA ratios. The sampling number was n ≥ 4 for all cases. “Control” represents the measurements of NIH 3T3 and P19 cells cultured for a day on polystyrene dishes in normal conditions. The ratio of settled cells after 15 min incubation prior to the AFM measurement (inset).

most of the cells remained detached for both NIH 3T3 and P19 cells. The settled cells appeared to keep a round shape on the coated dishes, in contrast to their morphology on polystyrene dishes, where the cells appeared to be more spread (Figure S3 of the Supporting Information). When the medium was exchanged to those for normal cell culture containing FBS, the cells on the BAM−BSA/BSA layer started to spread from the round shapes and became more adhesive; this cell spreading was further enhanced by addition of fibronectin. By examining DAPI-stained images, the cells appeared to be viable even after 6 h. (Figure S4 of the Supporting Information) To test the applicability of controlled adhesion using this system, we developed a method to measure the adhesion force C

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On a weakly adhesive dish, a single cell was successfully collected by capillary aspiration, indicating a potential use for single-cell manipulation. Adhered cells on 10% BAM−BSAcoated dishes were also successfully detached by rotational shaking of the dishes. As the detached cell ratio was clearly different, substrates with 10% and 100% BAM−BSA may be used as “detachable” and “stably adhesive” conditions in future applications. As the controlled adhesion of the substrate is applicable even for nonadherent cells, this method may be used for a wide variety of cell types. This transient cell-trapping technique has the merit of being simple and straightforward, as attachment/detachment can be controlled directly by adjusting the medium flow. This is very unique compared to other celltrapping techniques, such as temperature change, the use of special micropattern substrates or externally applied forces such as with optical tweezers, electric or magnetic fields, or dielectrophoresis (DEP).18−24 Being a simple method, the BAM−BSA/BSA-coating method can be combined with various other techniques used for cell manipulation, as it was compatible with the addition of fibronectin to enhance cell spreading and adhesion. Moreover, combination with patterning techniques to print BAM−BSA/BSA mixtures may lead to the production of novel cell arrays. Since this method is also useful for washing or transporting cells in microfluidics, it may assist in the integration of modules in μTAS devices.9,25 We believe this technique may contribute to the development of new technologies for biological diagnosis in the future.

Figure 3. A NIH 3T3 cell settled on a 10% BAM−BSA dish collected by microcapillary aspiration. Images are after different time intervals from the start of aspiration.

BSA dishes for 15 min. Here, the medium was not supplemented with EDTA, since the experiment could be completed within 10 min after cell settlement. For all cell types, most of the cells adhered during incubation, indicating that BAM−BSA-coated dishes could also allow the tethering of nonadherent cells, such as JM cells. This is in-line with what has previously been reported.14 Settled cells were exposed to the flow of the medium generated by the shaking of a rotary shaker at 100 rpm for 5 min. The force applied to the cells was estimated to be roughly 13 nN (Method S2 of the Supporting Information). As a result, a clear difference was observed in the percentage of released cell numbers between 10% and 100% BAM−BSA dishes for all cell types (Figure 4B). The adhesion of all cell types, including JM, was correlated with the BAM− BSA/BSA coating ratio.

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ASSOCIATED CONTENT

S Supporting Information *

BAM−BSA labeling ratio determination, method for the visualization of needle penetration, evaluation of the uniformity of the BAM−BSA/BSA-coated dish, morphology of cells on BAM−BSA/BSA-coated dishes, cell viability on 10% BAM− BSA dishes, estimation of flow shear force. This material is available free of charge via the Internet at http://pubs.acs.org.

4. CONCLUSIONS By coating dishes with a mixture of the adhesive BAM−BSA and the nonadhesive BSA, adhesion-controlled substrates were prepared. Force measurements using an AFM equipped with a hook needle revealed that the adhesion force of living cells to the substrate correlates with the BAM−BSA/BSA coating ratio.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +81-29-861-2445. Fax: +81-29-861-3048. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS 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).

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REFERENCES

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Figure 4. (A) 4 different types of cells (Hela, P19, NIH 3T3, and JM) were seeded on to BAM−BSA-coated dishes (35 mm in diameter). (B) After letting them settle for 15 min at 37 °C in a 5% CO2 incubator, the cells were exposed to a shear flow by use of a rotary shaker (100 rpm, 5 min) at room temperature and the percentage of released cells were calculated. D

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