Abstract
Eukaryotic cells in living tissues form dynamic patterns with spatially varying orientational order that affects important physiological processes such as apoptosis and cell migration. The challenge is how to impart a predesigned map of orientational order onto a growing tissue. Here, we demonstrate an approach to produce cell monolayers of human dermal fibroblasts with predesigned orientational patterns and topological defects using a photoaligned liquid crystal elastomer (LCE) that swells anisotropically in an aqueous medium. The patterns inscribed into the LCE are replicated by the tissue monolayer and cause a strong spatial variation of cells phenotype, their surface density, and number density fluctuations. Unbinding dynamics of defect pairs intrinsic to active matter is suppressed by anisotropic surface anchoring allowing the estimation of the elastic characteristics of the tissues. The demonstrated patterned LCE approach has potential to control the collective behavior of cells in living tissues, cell differentiation, and tissue morphogenesis.
INTRODUCTION
Living tissues formed by cells in close contact with each other often exhibit orientational order caused by mutual alignment of anisometric cells (1–4). The direction of average orientation, the so-called director
In this work, we present an approach to design tissues with a high degree of orientational order (up to 0.96) and a predetermined spatially varying director
We demonstrate that the structured LCE imposes a marked effect on the tissues, by controlling not only the alignment pattern but also the spatial distribution of cells, their density fluctuations, and even their phenotype as evidenced by different cells’ size and aspect ratio. The patterned LCE pins the locations of topological defects in tissues through anisotropic surface interactions and limits unbinding of defect pairs. The dynamics of defects in cellular monolayer at the patterned substrates is similar to the dynamics of active nematics formed by extensile units and allows us to estimate elastic and surface anchoring parameters of the tissue. The living tissues designed in our work resemble the tissues with topological defects grown by Saw et al. (5) at template-free isotropic substrates, with that difference that the defects in the LCE-patterned tissue are of a predetermined structure and appear at predetermined locations. Since the cells’ alignment patterns, topological defects in them, and even the defect cores control many important biochemical processes at microscale, such as action potential propagation (8) and apoptosis (5), our study opens the possibility to engineer platforms for the controlled patterning of tissues and their design for specific functions.
RESULTS
The LCE substrate is supported by a glass plate. To reduce surface roughness, the glass is covered with an indium tin oxide (ITO). The next layer is a photosensitive azo dye, the in-plane alignment of which is patterned by light irradiation with spatially varying linear polarization (21). This patterned azo dye layer serves as a template for the monomer diacrylate RM257 doped with 5 weight % (wt %) of photoinitiator I651 (fig. S1). After alignment through the contact with the azo dye, the monomer is UV-irradiated to photopolymerize the LCE substrate with a predesigned
(A) Digital holographic microscopy (DHM) texture of the LCE surface after contact with the aqueous growth medium. (B) Phase contrast-microscopy (PCM) texture of HDF cells growing on LCE substrates at 120 hours after seeding. Double-headed arrow represents
The orientational order of the elongated grains formed on a uniaxially aligned LCE,
The HDF cells are deposited onto the LCE substrate from the aqueous cell culture. When suspended in the culture medium, the HDF cells are round. Once an HDF cell sets onto the substrate, it develops an elongated shape with the axis of elongation along
The results on SHDF imply that the orientational order is caused mainly by the direct interactions of each cell with the LCE substrate. In the time interval of 24 to 168 hours, the cells migrate in the plane of the substrates with the speed ~102 μm/hour (movie S1). Development of orientational order in the HDF tissues is not associated with the evolution of the grainy texture of the substrate, since the latter, after the formation period of ~1 min, does not change over at least 14 days of observations (fig. S3).
The LCE substrates align not only the bodies of HDF cells but also their elongated nuclei (Fig. 1, D and E). This feature is important since orientation and shape of the nuclei affect many cell functions, such as protein expression, motility, metabolism, phenotype, and differentiation (23). The nuclei are elongated along the same direction as the cytoskeleton (Fig. 1D) and, apparently because of that, show a high order parameter reaching Snuclei = 0.88 ± 0.05 after 240 hours since seeding. The control ITO-glass substrates do not align cells, as there SHDF, Snuclei < 0.1 (Fig. 1, E and F, and fig. S4, B to D). Note here that after the cell monolayer is formed, further seeding and cell division might produce multilayered structures, but these are beyond the scope of the present study, in which the cells are fixed for fluorescent staining and analysis once the confluency is achieved.
Both aligned and unaligned assemblies of living cells exhibit high spatial fluctuations of number density (Fig. 1G), which is an attribute of out-of-equilibrium systems (24–26). In an equilibrium system, a certain area containing N objects would show fluctuations with SD ΔN proportional to N1/2. In contrast, the aligned tissue shows ΔN that grows somewhat faster than N1/2. To characterize the fluctuations, we followed (2, 26–28) and divided the tissue into small identical square areas and counted the number of nuclei in each of them, using fluorescent images such as the one in Fig. 1D. The calculations yield the mean number of nuclei 〈N〉 and the SD ΔN. Repeating the procedure for squares of different sizes, one obtains the dependency ΔN~〈N〉β, with β = 0.59 ± 0.03 for the aligned tissues and a similar β = 0.61 ± 0.03 for their unaligned counterparts (Fig. 1G). Both values are close to β = 0.66 ± 0.06 measured for a misaligned array of mouse fibroblast cells (2) and higher than the equilibrium value β = 1/2.
Elongation of grains along
(A) PolScope texture showing
The HDF cells self-organize into aligned assemblies that follow the preimposed director
The cells’ surface density σ(r) within the distance r < 60 μm from the +1/2 cores is substantially higher than near the −1/2 ones (Fig. 2E). The ±1/2 defects differ also in the characteristics of number density fluctuations. The fluctuations are stronger in the vicinity of −1/2 defects (β = 0.75 ± 0.05) as compared to +1/2 defects (β = 0.67 ± 0.05); in both cases, β is higher than the equilibrium value 1/2 (Fig. 2F). Here and in what follows, in the description of nonuniform patterns, all tissue properties, such as σ(r) and β, were determined by observing the fluorescently stained nuclei. Furthermore, β was calculated within square frames of a lateral size 800 μm, centered at the defect core.
The HDF cells alignment follows the patterned director of LCE that contains integer +1 defects of either pure splay (Fig. 3A, fig. S5C, and movie S5) or bend (Fig. 4A, fig. S5D, and movie S6). Azimuthal dependence of orientation θ of the HDF nuclei around −1 and +1 defects matches the predesigned
(A) PolScope image of
(A) PolScope image of
The LCE substrates impart a marked effect on HDF cells’ size and shapes. Since the cells are in contact with each other, the strong variations of σ’s at different LCE patterns, such as those in Fig. 4 (B, C, and E), translate into strong differences of cells’ size and shape. In Fig. 4 (B and E), the average values and their SDs, of the long (2a) and short (2b) axes of the HDF bodies approximated as ellipses, are 2〈a〉+1 = (68.2 ± 32.3) μm and 2〈b〉+1 = (26.2 ± 2.9) μm near +1 circular defect, whereas near −1 defect, the cells are significantly longer and slender, 2〈a〉−1 = (145.8 ± 38.6) μm and 2〈b〉−1 = (24.9 ± 5.1) μm. The shapes are analyzed within square frames of side length 800 μm, centered at the defect cores. The cells near the −1 defect are larger, of an average surface area 〈A〉−1 = 2.7 × 103 μm2 (calculated by dividing the area of 800 μm by 800 μm by the number of nuclei in it) and of a higher aspect ratio, 〈a/b〉−1 = 5.8 ± 2.7, as compared to their counterparts near the +1 defect, 〈A〉+1 = 1.8 × 103 μm2 and 〈a/b〉+1 = 2.6 ± 1.5 (Fig. 4, B and E, and fig. S8). These marked differences in the size and shape demonstrate that the LCE patterns influence the phenotype of HDF cells in tissues. It would be of interest to explore whether this functionality is unique to the LCEs, since Molitoris et al. (8) reported on lithographically aligned radial and circular +1 tissues but did not specified whether these patterns influenced the cells’ size or shape.
As demonstrated above in Fig. 2F, the exponent β characterizing the number density fluctuations of cells is different for the defects of charge −1/2 and +1/2. Thus, it is of interest to explore whether β depends on the particular type of deformations, splay and bend. Such a test is possible for +1 defect that can be of a splay (a radial aster) (Fig. 3D) or a bend (a circular vortex) (Fig. 4D) type. We find that the splay and bend do not produce a very strong difference in β, as β = 0.57 ± 0.04 for the radial +1 defect and β = 0.64 ± 0.03 for the circular +1 defect. In contrast, −1 defect in which splay and bend alternate produces slightly stronger fluctuations, with β = 0.68 ± 0.04 when −1 is paired with a radial +1 defect (Fig. 3D) and β = 0.70 ± 0.03 when paired with the circular +1 defect (Fig. 4D). We thus conclude that the number density fluctuations in tissues are influenced by the topological charge of the director patterns. To explore the issue in details, one would need to calculate the number fluctuations as a function of the radial distance to the defect core and the azimuthal coordinate for isolated defects of different topological charges; in these measurements, the patterned tissues should be of a much larger area than currently available.
In an equilibrium 2D nematic, the defects of an integer strength tend to split into pairs of semi-integer defects, since their elastic energy scales as ∝m2 (22). Both −1 and +1 defects in the HDF tissues split into two defects of an equal semi-integer charge separated by a distance d ≈ (200 to 500) μm. Inside the region r < d,
DISCUSSION
The data above demonstrate that the dynamics and proliferation of defects in patterned tissues can be arrested by the surface anchoring forces. In general, topological defects in sufficiently active matter tend to unbind and disorder the system (29, 30). The prepatterned tissue demonstrates that this defect unbinding can be prevented by interactions with an anisotropic patterned substrate that establishes a finite stationary separation distance d. Consider a pair of +1/2 disclinations that split from a parent +1 disclination at the substrate patterned as a +1 radial defect,
(A) A radial +1 defect in the underlying
According to Eq. 2, nonzero surface anchoring establishes a stationary finite separation of defects that is smaller in the radial geometry and larger in the circular geometry, in agreement with the experiment (Figs. 3, G and H, and 4, G and H). The difference in ds for circular and radial geometries is caused by the opposite directionality of the active force factive, as illustrated in Fig. 5. The experimentally determined value of this difference, Δds = factive/(2αW) ≈ 350 μm, allows us to estimate factive/W ≈ 140 μm. We also observe splitting of −1 defects. The resulting −1/2 defects can be considered in first approximation as inactive (29–31); thus, the separation distance is expected to be
The dependence of W on system’s parameters can be estimated by considering the nonflat profile of the substrates with a typical distance between the grains being λ = 10 μm and their height u0 = 0.1 μm (fig. S2). When a cell is aligned orthogonally to the elongated grains, it must bend around the grain. Assuming the substrate profile to be sinusoidal, u = u0 cos (2πx/λ), the bending energy density writes
In conclusion, we demonstrate that LCE substrates with photopatterned structure of spatially varying molecular orientation can be used to grow biological tissues with predesigned alignment of cells. Eukaryotic human tissues are designed with predetermined orientational patterns, structure, and location of topological defects in them. Besides the alignment, the substrates affect the surface density of cells, fluctuations of the number density, and the phenotype of cells as evidenced by different sizes and aspect ratios of the cells located in regions with different director deformations. In particular, higher density of cells is observed at the defect cores with positive topological charge, whereas the density is lower near the negative defects; cells are more round near positive defects and more elongated near negative defects. Anisotropic surface interactions between the tissue and the underlying LCE impose restrictions on the dynamics of topological defects in living tissues, preventing uncontrolled unbinding. The mechanism of cell alignment is rooted in swelling of the substrates upon contact with the aqueous cell culture medium; swelling produces anisotropic nonflat topography that follows the predesigned photopatterned director field of the LCE. We estimated the anchoring strength at the LCE-tissue interface and elastic modulus of the fibroblast tissue. The approach opens vast possibilities in designing biological tissues with predetermined alignment and properties of the cells, with precise location of orientational defects, which could lead to a controllable migration, differentiation, and apoptosis of cells. The proposed technique could be developed further by chemical functionalization of LCEs, by fabricating LCE substrates with dynamical topographies responsive to environmental cues, which would advance our understanding of the fundamental mechanisms underlying tissue development and regeneration. These studies are in progress.
MATERIALS AND METHODS
Substrate preparation and patterned alignment
We use ITO-coated glass slides of the rectangular shape and a size of 15 mm by 12 mm. The glass is cleaned in the ultrasonic bath of deionized water and small concentration of detergent at 65°C for 20 min. Next, we rinse the glass with deionized water and isopropanol and let it dry for 10 min, and after this, the glass is further cleaned inside the UV-ozone chamber for 15 min to remove any remaining organic contamination and improve the wettability properties. Immediately after that, the glass is spin-coated with 0.5 wt % azo dye SD-1 [synthesized following (33)] solution in dimethyl formamide, which serves as the photoaligning layer and annealed in an oven at 100 ° C for 30 min. The thickness of the azo dye layer is about 10 nm (34). For some samples, we use azo dye Brilliant Yellow (Sigma-Aldrich). Both azo dyes result in the same grain topography of LCE coatings and the same alignment quality of HDF cells. In contrast, replacement of the LCE with a polyimide PI2555 (Nissan) produced only flat topography (roughness on the order of 5 nm). We illuminate the glass with a metal-halide X-Cite 120 lamp through a linear polarizer to create uniform alignment (easy axis of alignment is perpendicular to the linear polarization of light) or through the special photomask (21), which creates high-resolution patterns of nonuniform director field. The illumination is performed for 5 min. The glass with azo dye coating is spin-coated with 6.7 wt % LC diacrylate monomer RM257 (Wilshire Technologies) and 0.35 wt % of photoinitiator I651 (Ciba Specialty Chemicals Inc.) in 93 wt % toluene and polymerize it with 365-nm UV light of a UVP-58 handheld lamp for 15 min (light intensity, 1.8 mW cm−2) at room temperature and ambient atmosphere. After full solvent evaporation, the concentration of photoinitiator I651 becomes 5 wt %. If the content of I651 is increased to 10 wt %, then the resulting LCE coating shows the same surface grainy topography when in contact with the cell culture medium. We calculated the thickness of the LCE layer from the average optical retardance Γ = 40 nm measured with PolScope in cells with a uniform planar director field. Since the birefringence of RM257 is about Δn ≈ 0.2 (35), the thickness of the LCE layer is Γ/Δn = 200 nm. The substrate is placed into a petri dish, additional sterilization and cleaning is done inside an UV-ozone chamber, and then the HDF cells are seeded onto the substrate and allowed to adhere, grow, and multiply on it. We also tested LCE substrates with fibronectin extracellular matrix coating (fig. S10). The fibronectin-coated substrates were prepared by immersing LCE substrates into the solution of 1 wt % fibronectin (Thermo Fisher Scientific) in deionized water and incubated for 2 hours. Fibronectin is an agent universally used for a better adhesion of HDF cells to substrates. In combination with LCE substrates, fibronectin coatings produced similar results as LCE coatings without this agent; thus, in the main text, we present the data for fibronectin-free substrates.
Cell plating
HDF cells were purchased from American Type Culture Collection (catalog no. PCS-201-010) and maintained at 37°C with 5% CO2 and >90% humidity inside the incubator. Passages 1 to 6 are used in the experiment, and no significant difference in the behavior of different passages is recorded. Cell culture medium is composed of Dulbecco’s modified Eagle’s medium (high glucose) supplemented with 10% fetal bovine serum (FBS) (Clonetics), GlutaMAX, and penicillin/streptomycin (both from Thermo Fisher Scientific). The cells were plated at the surface density 3.3 × 107 m−2 (fixed for all experiments) onto the substrates placed in a Petri dish made of a sterilized glass [to allow for the polarized optical microscopy (POM) imaging].
Immunocytochemistry
F-actin cytoskeleton and nuclei in HDF cell cultures are immunofluorostained. Cell cultures are fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 in phosphate buffer saline (PBS) for 25 min, and nonspecific enzymatic activity was blocked with 5% FBS in PBS for 20 min. Cultures are then incubated with Alexa Fluor 488 Phalloidin (1:100 dilution, A12379, Invitrogen) for 30 min and 4′,6-diamidino-2-phenylindole (DAPI; 1:5000 dilution, D1306, Invitrogen) for 10 min.
Microscopic observation
The cells are examined under inverted phase-contrast microscopes AmScope AE2000 or Leica DH (equipped with AmScope MU-2003-BI-CK or Pixelink PL-P755CU-T cameras, pair of polarizers, and 4×/Ph0 or 10×/Ph1 objectives) without temperature and environmental control for a short period of time (not more than 5 min) every 6 or 24 hours and kept inside the incubator for rest of the time until cells reached confluency. At that point, cells’ nuclei and cytoskeleton actin filaments are stained with the fluorescent dyes (see the “Immunocytochemistry” section) and observed under the fluorescent microscope Nikon TE-2000i with sets of fluorescent cubes that allow excitation of different fluorescent dyes. The fluorescent data are recorded with an Emergent HS-20000C camera with 10× and 20× long–working distance objectives. The surface characterization is performed using atomic force microscopy (AFMWorkshop), scanning electron microscopy (Quanta 450 field emission gun environmental scanning electron microscope), and digital holographic microscopy in a reflection mode (Lyncée Tec digital holographic microscope R-1000). Imaging with the fluorescent microscope Nikon TE-2000i allows us to visualize elongated shapes of the cell nuclei and to determine Snuclei as described below. PolScope microscopy is used for birefringent materials, such as orientationally ordered LCE, and allows one to determine the local orientation of the in-plane optic axis (which coincides with the director
Image analysis
Images are processed in open-source software package Fiji/ImageJ2 (37), further analysis is performed with the custom-written code in MATLAB (MathWorks) and Mathematica (Wolfram) proprietary packages. The local cell orientation is obtained from the phase-contrast microscopy images and computed using OrientationJ Fiji/ImageJ2 plugin (38), which calculates local orientational tensor using a finite-difference intensity gradient method within the small interrogation window. The minimum window size of 20 μm was chosen to be of the order of typical isolated HDF cell width on the LCE substrates. The long-axis orientation and position of the cells’ DAPI-stained nuclei is obtained from fluorescent microscopic images. The orientation and intensity-weighted center of mass of each nucleus is calculated with Extended Particle Analyzer BioVoxxel Fiji/ImageJ2 plugin (39). Analysis of the azimuthal distribution of orientation and radial distribution of HDF cell nuclei, number density, and number density fluctuations is performed using a custom-written program in MATLAB package. Radial dependence of the surface area density σ(r) is calculated as the ratio between number of nuclei and the area of the radial annulus of width 10 μm at the given radius r from the center of defect cores in the LCE substrate.
Determination of scalar order parameters
To determine the scalar order parameter of elongated LCE grains (SLCE), HDF cells body (SHDF), and elongated HDF nuclei (Snuclei), we first calculate the tensor order parameter Qij = 2〈uiuj〉 − δij (22). Here, uiuj is the dyadic product of unit vector
Surface anchoring energy calculation
To obtain the total surface free energy of the array of HDF cells on LCE, we integrated the surface anchoring energy per unit area
SUPPLEMENTARY MATERIALS
Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/20/eaaz6485/DC1
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REFERENCES AND NOTES
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