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. 2019 Nov 21;4(23):20223–20229. doi: 10.1021/acsomega.9b02522

Single Polar Cell Trapping Based on the Breath Figure Method

Xixi Wu †, Tao Liu ‡, Su Gao §, Shuangshuang Chen †,*, Qinghua Lu †,§,*
PMCID: PMC6893950  PMID: 31815223

Abstract

graphic file with name ao9b02522_0008.jpg

The ability to research individual cells is important for various biological studies. Currently reported biointerfaces for single-cell analysis can only trap individual cells in random morphologies. Cell polarity is a key factor in cellular functions, and the study of single-cell polarity can facilitate an understanding of cancer metastasis and stem-cell differentiation. For single polar cell trapping, anisotropic honeycomb-structured films were prepared. Elastic poly(1,2-butadiene) honeycomb films with ordered hexagonal pores were first prepared via the breath figure method. Subsequently, the films were subjected to mechanical stretching and fixed via photo-cross-linking under UV light irradiation. This stretched honeycomb structure was then transferred to a polystyrene surface. The resultant anisotropic porous films exhibited excellent capacity for single-cell trapping. Besides contributing to the physical spatial confinement of cells, the trapped single cells exhibited orientation in different polarities. The single polar cell array provided a novel platform for fundamental biological research.

Introduction

Conventional cell analysis methods include enzyme-linked immunosorbent assay, western blotting, and real-time polymerase chain reaction, which have been extensively applied in biological research.1 These analysis methods are used to detect cellular processes and responses by measuring a group of cells and producing an average result, based on the assumption that there is no difference between individual cells in cell populations. However, it is very difficult to obtain sensitive signals from an overall analysis of many cells. Specifically, conventional population-based cellular analysis cannot detect cellular heterogeneity.2 Furthermore, cell-to-cell interactions have been proven to influence cellular behaviors, so investigating these interactions can provide insights into real cell–material relationships. For example, the differentiation of stem cells on two-dimensional culture platforms is usually realized by adding growth factors, which might also be influenced by intercellular communications. Cells with multiple biological interactions cannot be used to determine intrinsic cellular principles. Recently, cellular heterogeneity has attracted increasing attention to cell-to-cell differences in response to internal or external stimulation. Given this demand, single-cell analysis technology has rapidly developed.

In the analysis of single cells, to prevent interference from cell–cell interaction, trapping or immobilizing individual cells is a fundamental task. Numerous techniques have been developed to create microarrays that can trap single cells, based on the guidance of optical, magnetic, electrical, centrifugation, ultrasonic, pressure, and hydrodynamic forces.3−8 Among these techniques, microfluidic arrays have been most commonly applied for their ability to simultaneously immobilize many single cells and enable in situ observation over time.9 However, their complicated fabrication and the associated expense of photolithography and soft lithography have hindered the commercialization of these chips.10,11 In addition, most of the trapped cells are always kept in round traps, which might influence their cellular functions.

Cell polarity is a common feature of many different cell types and is an essential factor in differentiating and determining the function of most cells.12,13 Cell polarity refers to the concentration of certain cytoplasmic components in a certain spatial order, which results in a concentration gradient of various cell contents. Cell polarity is very important for normal cell function and plays a key role in many biological processes, including cell differentiation, cell migration, cytokinesis, and tissue and organ formation.14,15 Loss of polarity is also related to disease states such as cancer.16−18 However, such polarization for single-cell analysis has remained unclear. Herein, it is urgent to develop single polar cell trapping techniques to study locally polarized cell behaviors.

In this study, we employed the breath figure method to fabricate porous films with an average pore diameter of 18 μm.19 After fabrication, the films were physically stretched, photo-cross-linked, and replicated to produce stable anisotropic holes with a tunable aspect ratio (Scheme 1). Using the patterned substrates, the effects of size and aspect ratio on cell pseudopodia and polarization were investigated. To the best of our knowledge, there have been no previous reports on the control of polarity when performing single-cell trapping and analysis. The simple method proposed in this work provides a novel platform for the analysis of single polar cells in biological research.

Scheme 1. Honeycomb-Patterned Polybutene (PB) Films Were Prepared Using a Typical Breath Figure Approach, Followed by Mechanical Stretching of the Elastic PB Films.

Scheme 1

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The honeycomb films with a series of stretched ratios were then cross-linked and fixed via UV irradiation. Subsequently, the anisotropic honeycomb structures were applied for single-cell trapping.

Results and Discussion

Preparation of Stretched Honeycomb Surface

PB honeycomb films have been reported by Shimomura et al., which were incorporated with an amphiphilic polymer as emulsifier to balance the uniformity.20 However, the diameter of the pores in that study was only about 6 μm, which was too small for trapping cells. Meanwhile, the used amphiphilic copolymer was hard to be obtained. For larger hole size, pure hydrophobic PB was used for honeycomb films. According to the principles of the breath figure technique, morphological characteristics such as pore diameter and depth can be easily modulated by changing the preparation conditions.19 In this study, the preparation conditions were optimized first by changing the PB concentration, environment humidity, and airflow (Figure S1). As shown in Figure 1a,d, a relatively ordered honeycomb structure can be obtained on PB film with a diameter of 18 ± 2.0 μm at an airflow rate of 50 mL/min and at 65% humidity. As well known, the diameters of most studied eukaryotes range from 10 to 20 μm. Therefore, the honeycomb pores provide size-matched cavities for trapping cells. Furthermore, the depth of the honeycomb pores also plays a critical role in trapping cells, limiting cell motion, and preventing intercellular interactions. Cross-sectional scanning electron microscopy (SEM) images of the honeycomb films reveal that the depth of the original holes was approximately 6 ± 0.35 μm (Figure 1g–i).

Figure 1.

Figure 1

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(a–c) Optical images, (d–f) SEM images, and (g–i) cross-sectional SEM images of honeycomb structures with various stretching ratios (1:1.0, 1:1.5, and 1:2.0), with inserted figures showing simplified models. (j) Average transverse and longitudinal lengths of the stretched honeycomb. (k) Ratios of longitudinal and transverse lengths on different stretched surfaces. (l) Average depths of honeycomb holes on corresponding surfaces.

For the polar cell trapping, anisotropic structures were prepared by physical stretching. Flexible PB films were stretched to lengths 1.5 times and 2.0 times the original films and then fixed with glue onto glass slides (Figure S2). Subsequently, the PB films were UV-cross-linked and transferred to polystyrene (PS) (Figure S3). The stretched honeycomb structures exhibited an anisotropic shape with the larger axis along the stretching direction. Round holes were obviously changed to thin elliptical shapes after being stretched to twice their original length (Figure S4). The precise diameters along the transverse and longitudinal axes were quantitatively measured and shown in Figure 1j. During the stretching, there was an obvious increase in the longitudinal diameter but a slight decrease in the transverse diameter. The anisotropy of the honeycomb structures was evaluated by the ratio of the longitudinal to transverse diameters (Figure 1k), which exhibited a linear relationship with the degree of stretching. Therefore, the degree of anisotropy can be easily modulated by directly changing the stretching length. Furthermore, the depth of the honeycomb pores was nearly maintained, thus providing physical barriers to intercellular communications (Figure 1l).

Wettability of Anisotropic Honeycomb Surfaces

The surface wettability was determined by two factors, namely chemistry and morphology. As mentioned above, the stretched honeycomb films exhibited anisotropic morphologies, which might lead to anisotropic wetting behavior. As a proof of concept, the water contact angles (WCAs) were measured along the transverse and longitudinal axes. The WCA of the original honeycomb film shown in Figure 2 was about 70°, which is slightly hydrophobic because of the inherent hydrophobicity of PS and the surface pore structure. After being stretched, the WCA along the transverse axis increased, whereas that along the longitudinal axis decreased. This is because the transverse diameter of the elliptical pores was smaller than that of the longitudinal diameter, resulting in higher roughness and more energy barriers in transverse direction for wetting.21 The anisotropic wetting behavior of anisotropic structures might influence cellular adhesion and deformation (see Figure 2).

Figure 2.

Figure 2

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WCAs of various stretched honeycomb films. Anisotropic wettability was evaluated in the longitudinal and transverse directions.

Biocompatibility of Prepared Stretched Honeycomb Surfaces

We evaluated the viability of cells on original and anisotropic honeycomb films. Both L929 cell and 293t cell suspensions at a concentration of 105 cells/mL were seeded onto the surfaces. After incubation for 24 h, the nucleus of trapped cells was stained with live/dead (AO/EB) assay. The nuclei of all adhered cells were stained with green fluorescence indicating good viability. The viability of cells was approximately 100%. No difference was observed between these films, indicating that both original and anisotropic honeycomb films were biocompatible for cell adhesion. Under reflection mode, the honeycomb walls portrayed a vivid boundary of each holes. The captured cells can be obviously located in honeycomb holes individually, which proved that the honeycomb films fabricated via the breath figure method can be used for single polar cell trapping (Figure 3).

Figure 3.

Figure 3

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Fluorescent images of live/dead (AO/EB) stained cells (L929 and 293t) on anisotropic surfaces after incubation for 15 and 24 h. The scale bar is 80 μm.

Single-Cell Trapping

Honeycomb structures with ×1.0, ×1.5, and ×2.0 anisotropies and an original diameter of 18 μm were used to study cell-trapping behavior. To visualize the trapping process, the cells were prestained with live fluorescent calcein-AM. The cell immobilization on the honeycomb films might be influenced by various factors, such as the settle time and cell density. Cell adhesion is reported to occur quickly, within 40 min after cell–substrate contact.22 Here, a cell settle time of 1 h was selected, which was long enough for cell adhesion to occur, and a series of concentrations ranging from 104 to 106 cells/mL were applied to optimize the cell-trapping experiment. The percentage of honeycomb holes that contained cells is referred to as cell occupancy. The percentage of individually immobilized cells among all the trapped cells is referred to as the single-cell ratio (efficiency), which can be used to evaluate the cell-trapping capacity on different substrates.

Figure 4a–c shows the effect of cell density on trapping efficiency. When seeded with a low concentration of cells, nearly all the cells were individually immobilized in honeycomb holes, with a high single-cell trapping efficiency (Figure 4a). However, because of the lack of enough cells, cell occupancy was relatively low. Therefore, the overall trapping amount and single-cell efficiency were unacceptable. By increasing the cell concentration to 105 cell/mL, there was an obvious increase in cell occupancy (Figure 4b,d). At the same time, the single-cell ratio was well maintained when compared with that shown in Figure 4a. With respect to the concentration of 106 cells/mL, the cell occupancies of all the honeycomb films were improved to approximately 80%, but the single-cell ratio declined significantly (Figure 4e). The unstretched honeycomb films maintained a single-cell ratio above 60%, which might be attributed to the spatial barrier. The longitudinal length of the honeycomb holes on the ×2.0 anisotropic film increased to 35 μm, which might accept two or more cells. Therefore, several cells may get trapped in a stretched honeycomb cavity, which would result in a low single-cell ratio. Taking both cell occupancy and the single-cell ratio into consideration, the concentration of the seeding-cell suspension was optimized to be 105 cells/mL.

Figure 4.

Figure 4

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Fluorescent images of calcein-AM-marked L929 cells on isotropic and anisotropic honeycomb films. The concentrations of cell suspensions were regulated from 104 (a), to 105 (b), and 106 cells/mL (c). The scale bar is 100 μm. Cell occupancy on different honeycomb films (d) and corresponding single-cell trapping efficiency (e).

Polarity of Single Trapped Cells on Anisotropic Honeycomb Films

Cell morphology is largely influenced by the surrounding microenvironments, including stiffness, roughness, curvature, and charge.23 The SEM images vividly show the cell polarity in anisotropic holes after culturing for 8 h (Figure 5a,b). Because of spatial confinement, 293t and L-929 cells maintained their spherical shapes on the original honeycomb surfaces. However, significant changes in cell shape were observed in the cells in the stretched anisotropic honeycomb holes. These cells spread along the direction of the longitudinal axis and had more substantial pseudopods. The one-axis elongation in the ×2.0 anisotropic hole, namely, its polarity, was larger than that in the ×1.5 anisotropic hole. This is speculated to be due to a synergistic effect between the intrinsic cell polarization and anisotropic cue from the wettability and the confined space and roughness of the stretched honeycomb surface. A large number of studies have reported the effect of micro–nano structures on cell morphology and have proved that cells can transmit external environmental information inside cells through receptors on their pseudopodia and that the cytoskeleton can make adaptive adjustments that result in changes in cell morphology.24 However, this is the first study of the polarization of a single cell inside a cavity, which differs from the behavior of the cell population. This research is significant for the study of single-cell behavior and single-cell analysis.

Figure 5.

Figure 5

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SEM images of 293t (a) and L-929 (b) on isotropic and anisotropic honeycomb films after culturing for 8 h. The scale bar is 10 μm. Confocal fluorescent images of immunostained 293t (c) and L-929 (d) cells on anisotropic honeycomb films after culturing for 8 h. The scale bar is 15 μm. In the images, blue is the nucleus stained by 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI), green is the adhesion protein stained by fluorescein isothiocyanate (FITC)-labeled antivinculin, and red is the cytoskeleton stained by rhodamine phalloidin.

To further investigate the individual cell polarity in anisotropic honeycomb surface, immunofluorescence staining was conducted to trace cytoskeleton and distribution of focal adhesion proteins. The red actin microfilament shows the integral shape of the cell in accordance with SEM results, that is, a cell in the original hole had a round spherical shape, whereas it was elongated in the anisotropic cavity. Vinculin, a component of focal adhesion proteins, plays a significant role in cell adhesion, cell migration, and cell deformation.25−27 The vinculin expression in the cell trapped in the original honeycomb structure was weak and tended to aggregate in the middle of the cell to some extent. With an increase in the anisotropy, the distribution of vinculin occurred more and more around the two cell polarities. In particular, the intensive green fluorescence at the front ends of the pseudopods can be observed from cells on the ×2.0 anisotropic surface, which indicates polar cell growth (Figure 5c,d).

Growth in the polar cells was further demonstrated by the cell spreading area and aspect ratio with time (Figure 6 and S5). In the earlier period of cell adhesion (4 h), the spreading area and aspect ratio of cells on different substrates were similar. After culturing for 6 and 8 h, the cells trapped on isotropic surface still exhibited nonpolar morphologies and were slightly spread. However, the cell spreading area on the anisotropic surface increased sharply, accompanied by an increase in the aspect ratio. Focal adhesion kinase (illustrated by vinculin) was also found to be distributed along the longitudinal direction (Figure 5c,d), and the cytoskeleton was also regulated and arranged along the longitudinal direction. Therefore, single cells trapped in anisotropic honeycomb cavities exhibited polar growth. In addition, the prepared anisotropic honeycomb films can also be applied for analyzing cancer cell (illustrated by HeLa cell in Figure S6), demonstrating a common platform for single polar cell analysis.

Figure 6.

Figure 6

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Statistical analysis of spreading area (a) and polarity (b) of individual L929 cells after being trapped in anisotropic honeycomb films.

Conclusions

In conclusion, ordered honeycomb-structured films with a cell-matched diameter were prepared via the breath figure method. The honeycomb films were stretched to obtain cavities with ×2.0 and ×1.5 anisotropy. The prepared honeycomb films exhibited good single-cell trapping capacity via the separation and locking of individual cells in each honeycomb hole. Because of the spatial confinement, the individually trapped cells were observed to grow and develop polarity inside the stretched honeycomb cavities. Single-cell trapping in honeycomb films and polar cell growth in anisotropic cavities might have a broad applicability in single-cell studies for biological research.

Experimental Section

Materials

Chloroform and ethanol (analytical purity) were purchased from Sinopharm Chemical Reagent Co. Ltd., Shanghai, China, without further treatment. The polydimethylsiloxane (PDMS) precursor (184) was obtained from DOW Chemical Co., USA. Poly(1,2-butadiene) (PB) with an average Mw of 200 000–300 000 g/mol was purchased from Sigma. PS (average Mw 250 000 g/mol) and 1H,1H,2H,2H-perfluorodecyltrichlorosilane were supplied by Acros. 293t and L-929 cells were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Science. Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were supplied by Gibco. Other reagents for cell culture and dye for cell staining were all purchased from Beyotime Biotechnology.

Characterizations

The morphologies of the prepared honeycomb substrates were imaged on a FlexSEM 1000 (Hitachi, Japan). The honeycomb films were also submitted to optical microscopy (Olympus, China) using optical transmission mode. The WCAs of patterned surfaces were measured on OCA 10 (DataPhysics, Germany), and the measurements were then recorded along and against the stretching orientation using 4 μL of water droplets for each test. All fluorescent images were obtained using an inverted fluorescence microscope (DMi 8, Leica, Germany) under optical reflection mode.

Fabrication of Honeycomb Scaffolds

According to the method described in the literature,19,20 PB (250 mg) was dissolved in chloroform (10 mL) and the solution was then cast onto a glass substrate and placed in a self-prepared chamber with variable humidity. The honeycomb structure was formed on the PB surface at a designed level of humidity using the breath figure approach. To obtain an anisotropic structure, the films were then stretched using a tensile machine (HY-0350). The stretched PB films were then fixed by photo-cross-linking under UV irradiation at a power level of 18 W for 12 h.

To obtain a stable substrate, the patterns on PB were replicated by performing two transfers to PS substrates that are frequently used for cell culture, as shown in the schematic in Figure S4. The stretched PB films were first functionalized with a monolayer of 1H,1H,2H,2H-perfluorodecyltrichlorosilane by vapor phase deposition for 12 h. The PDMS precursor with a curing agent (the ratio of PDMS to curing agent was about 10:1) was then cast onto the functionalized PB substrate. After curing the PDMS, the PS chloroform solution (0.1 g/L) was cast onto the PDMS template to obtain PS films with a stretched honeycomb structure.

For more information of substrates, the parameters including diameter and depth of anisotropic honeycomb films were statistically measured on more than six SEM images using Nanomeasurer software.

Cell Trapping and Culture

To trap single cells, the PS honeycomb films were first rinsed three times with phosphate-buffered saline (PBS) and then sterilized using medical ethanol and UV irradiation. Before performing the trapping test, the biocompatibility of the films was evaluated by live/dead (AO/EB) dual staining and seeding the cell suspension at a concentration of 1 × 105 cells/mL. The cells on the elongated PS honeycomb films were then cultured with DMEM (containing 10% FBS and 1% penicillin–streptomycin) in a humidified cell incubator with a 5% CO2 atmosphere for 5, 10, 15, and 24 h.

After the cell compatibility test, single-cell trapping was performed at different cell concentrations (104, 105, and 106 cells/mL, respectively), using epithelial cells (293t) and fibroblasts (L929) as modules. The cell suspensions were seeded onto the elongated honeycomb PS films for 1 h. Then, unattached cells were removed and rinsed using PBS, and a fresh cell culture medium was added to continue the culture process. Live cells were then stained using dye (calcein-AM) to make the captured cells visible and enable calculation of the single-cell capture efficiency on substrates with different stretching ratios. The polarity of the single cells that had been trapped in the honeycomb pores were determined and recorded at 4, 6, and 8 h, respectively.

Immunofluorescence Staining and Cell Morphology

To obtain more detailed information about the adhered cells, the cytoskeleton and adhesion protein expression were imaged via immunofluorescence staining. The trapped individual cells on the honeycomb films were fixed with a 4% paraformaldehyde (PFA) solution for 10 min. Then, the cells were rinsed with PBS three times and permeabilized with an enhanced immunostaining permeabilization solution (Beyotime Biotechnology) for 10 min. To resist nonspecific adhesion, the cells were blocked with bovine serum albumin (BSA) for 30 min. Then, an FITC-labeled antivinculin monoclonal antibody (1 μg/100 μL in PBS and blocked by 0.5% BSA) was used to immune-stain the distribution of cellular adhesion proteins by incubating the cells for 45 min. The remaining antibody solution was taken away and washed with PBS four times (5 min each) to remove unbound antibodies. Rhodamine-labeled phalloidin (100 nM in PBS with 0.5% BSA) was then added and incubated for 45 min, after which the unbound phalloidin was rinsed with PBS. Finally, DAPI was applied to observe the morphologies of the cell nuclei. After sealing, the stained cells were imaged using a confocal laser scanning microscope (Zeiss LSM880, Carl Germany).

SEM was also used to obtain morphological information about the trapped cells. The cells were primarily fixed using 5% PFA, and these fixed cells were treated with gradient ethanol solutions (30, 50, 70, 95, and 100%; each concentration for 15 min) to remove water from the samples. The fixed cells were dehydrated in a dryer at room temperature. After being sputtered with a conductive ion layer, the cell morphologies were observed using the FlexSEM 1000 (Hitachi, Japan).

Statistical Analysis

The experimental data were collected at least three times unless otherwise stated. The mean values were presented as ±standard deviation and tested using a one-tailed heteroscedastic t-test. Differences were statistically significant at p < 0.005.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (21704076 and 51573089). The authors also appreciate the support of the “China Postdoctoral Innovation Talent Project” (BX201700171) and the China Postdoctoral Science Foundation (188682).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.9b02522.

  • Optimized honeycomb structures, cross-sectional SEM image of honeycomb films, scheme of replication from PB to PS cellular morphologies after culture for 4 and 6 h, and HeLa cell trapping (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao9b02522_si_001.pdf (723.3KB, pdf)

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Supplementary Materials

ao9b02522_si_001.pdf (723.3KB, pdf)

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