Proliferation of Cells with Severe Nuclear Deformation on a Micropillar

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Biological and Environmental Phenomena at the Interface

Proliferation of Cells with Severe Nuclear Deformation on a Micropillar Array Ruili Liu, Xiang Yao, Xiangnan Liu, and Jiandong Ding Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03452 • Publication Date (Web): 04 Dec 2018 Downloaded from http://pubs.acs.org on December 5, 2018

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Proliferation of Cells with Severe Nuclear Deformation on a Micropillar Array Ruili Liu, Xiang Yao, Xiangnan Liu, Jiandong Ding1 State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Fudan University, Shanghai 200438, China

Abstract Cellular responses on a topographic surface are fundamental topics about Interfaces and Biology. Herein, a poly(lactide-co-glycolide) (PLGA) micropillar array was prepared and found to trigger significant self deformation of cell nuclei. The time-dependent cell viability and thus cell proliferation was investigated. Despite of significant nuclear deformation, all of the examined cell types (Hela, HepG2, MC3T3-E1 and NIH3T3) could survive and proliferate on the micropillar array, yet exhibited different proliferation abilities. Compared to the corresponding groups on the smooth surface, the cell proliferation abilities on the micropillar array were decreased for Hela and MC3T3-E1 cells, and did not change significantly for HepG2 and NIH3T3 cells. We further found that whether the proliferation ability changed or not was related to whether or not the nuclear sizes reduced in the micropillar array, and thus the size-deformation of cell nuclei should, besides shape-deformation, be taken into consideration in studies of cells on topological surfaces.

Keywords:

Proliferation, Nuclear deformation, Polymeric materials, Surface

patterning, Micropillar array, Cell-material interactions

Corresponding author. Tel.: 86 21 31243506. E-mail address: [email protected] (JD Ding). 1

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INTRODUCTION Cell-material interactions constitute important fundamental topics in the fields of biomedicine

and

biomaterials.1-9

Surface

topography

and

physicochemical

environment influence the cellular behaviors profoundly at the cell-substrate interface.10-11

With

the

development

of

surface

patterning

techniques,12-19

appropriate microscale and nanoscale features have been found to influence cell behaviors without necessarily destroying the cellular biochemical environment. Topological controls of various cellular responses, including cell adhesion, proliferation, migration and differentiation, have been reported in recent years.20-37 The underlying mechanisms of the cellular responses to topological features have been proposed recently, such as the discovery of the key proteins of the membrane curvature.38-40 A micropillar array as a type of quasi-three dimensional cell culture substrates affords a unique biophysical cue to tune cell-material interactions, and has got to be a promising platform for biomedical research such as drug screening, wound healing etc.41-49 A quasi-three or three dimensional cellular microenvironment is valuable as physiological or pathological models to gain fundamental understandings of biology, diseases or more accurate prediction of drug responses.50-54 The cell nucleus as the largest cell organelle is also affected by the micropillar topography at the subcellular level. Nuclear deformation induced by micropillar arrays with appropriate dimensions has been found recently.55-61 Severe self deformations of cell nuclei of osteosarcoma-derived cell lines and bone marrow stromal cells were observed on the micropillar array by Anselme group and our group.55, 57 Appropriate dimensions such as a sufficient height and an appropriate gap between the pillars are required for the micropillar array to induce the severe cell nuclear deformation.57-58 The geometry of cell nuclei has further been considered to be a new cue to control the lineage commitment of stem cells.59 The partial recovery of the nuclei of mesenchymal stem cells (MSCs) was also discovered, which accounted for the overshoot phenomenon of the nuclear deformation.60 Very recently,


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we reported a cell type dependence of nuclear response on a micropillar array: epithelial-like cells exhibited a more severe nuclear deformation than fibroblast-like cells.61 The nuclear-size dependence of self deformation of cell nuclei on the micropillar array was also discovered.61 The cell nucleus possesses a tensegrity structure with chromatin fibers and a surrounding scaffolding of the nuclear matrix.62 External forces on the nucleus may modify the nuclear shape, change the chromatin organization, and subsequently induce the modification of gene expression, which turns out to affect the cellular behavior. Decondensation of chromatin has been proved to induce an increase of cell nuclear deformability.63 Although modification of cellular behaviors owing to nuclear deformation has been observed such as the lineage commitment of stem cells on the micropillar array,59 some other important cellular responses to the nuclear deformation, such as cell proliferation ability, are less understood and much required to be revealed.

Figure 1. Schematic presentation of self deformation of cell nuclei on micropillars and comparison of corresponding cell proliferation ability of different cell types.

Herein we address the questions, as schematically presented in Figure 1. In order to answer the questions, we prepared, using a micropit array as a template, a poly(D,L-lactide-co-glycolide) (PLGA) micropillar array of an appropriate dimension to trigger significant self deformation of cell nuclei. The proliferation ability of human cervix epithelial carcinoma cells (Hela) on the micropillar array was determined and compared with that on the smooth surface. A decreased proliferation ability was detected for Hela cells on the micropillar array. Other three types of mammalian cells were further examined, including human hepatoma cells (HepG2), mouse osteoblastic cells (MC3T3-E1), and mouse embryo fibroblast cells (NIH3T3).


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The time dependent viability and thus the proliferation ability of cells with deformed nuclei

were

quantitatively

characterized

by

a

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the DNA content determination. Behaviors of cells on the PLGA micropillar array were compared with those on a flat or smooth PLGA surface. Considering that nuclear size influences the condensation state of the chromatin and the DNA synthesis,64-66 we also quantified nuclear sizes of the cells on the micropillar array and checked its relation with the change of proliferation ability.

MATERIALS AND METHODS Micropillar Fabrication. PLGA is a very useful biodegradable polymer.67-71 PLGA85/15, with number average molecular weight 3.59×105 and molar mass dispersity 1.72, containing 85 wt% lactide (LA) and 15 wt% glycolide (GA) units, was purchased from Purac Inc. Netherlands. A micropit silicon wafer (ordered from Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences) was used as the template to prepare the copolymer micropillar array. The PLGA solution in dichloromethane (5 wt%) was poured onto the silicon wafer. After the solvent was evaporated, we peeled off the PLGA micropillars, and observed the micropillar array with scanning electron microscopy (SEM, VEGA TS 5136MM, TESCAN) and confocal laser scanning microscopy (Nikon, C2+, Japan). Cell Culture. Four cell types were examined, including Hela, HepG2, MC3T3-E1 and NIH3T3. All of the cells were purchased from Cell Bank, Type Culture Collection, Chinese Academy of Sciences. All of the cells were cultured in high-glucose Dulbecco‟s modified Eagle medium (DMEM) with 10% fetal bovine serum. Glutamine (2mM), penicillin (100 units/mL), and streptomycin (0.1 mg/mL) were added into culture media. We sterilized the PLGA films with or without micropillared surfaces with 75% alcohol. Cells were cultured on the sterilized PLGA films at the density of 20 cells per mm2 (1×104 cells per well) in 12-well tissue culture


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plates (TCPs). Cells were cultured in a humidified incubator at 37 oC with 5% CO2 atmosphere. Cell Staining. A warm phosphate buffer saline (PBS) solution was used to wash cells. Then the adherent cells were fixed in 4% paraformaldehyde for 10 min, and permeabilized with a 0.1% v/v Triton X-100 for another 10 min. In order to label the filamentous actins (F-actins), we incubated the cells with 1 μg/ml Phalloidin-TRITC (Sigma) at room temperature for 30 min. We labeled cell nuclei with 5 μg/ml 4‟,6-diamidino-2-phenylindole (DAPI, Sigma) for 10 min after rinsing the samples with PBS. After the cells were rinsed with Milli-Q water, all the stained samples were observed with an inverted microscope (Axiovert 200, Zeiss) mounted with CCD (AxioCam HRC, Zeiss). Quantification of Shapes of Cells and Cell Nuclei. The shape index (SI), which is defined as 4πS/l2, was used to quantify the extent of nuclear deformation based on two dimensional micrographs of cell nuclei. The areas (S) and perimeters (l) of both cell bodies and cell nuclei were measured using Image-J software (freely available at http://www.nih.gov) based upon fluorescence micrographs of F-actins and nuclei, respectively. The index is 1 for a perfect circle and 0 for a straight line. A nucleus with SI < 0.8 was considered a deformed one.59 Nuclei of at least 300 cells were measured for each group. Determination of Volume of Cell Nuclei. To confirm the three-dimensional morphology of a nucleus, we captured confocal fluorescence image slices of the cell nucleus in the range of nuclear thickness at 0.5 μm intervals, using a confocal system (C2+; Nikon, Japan) with a multicolor fluorescence system. The cell volume was obtained by summation of a stack of serial sections, as schematically presented in Figure S1. If Sn and Sn-1 indicate the nuclear areas of the n and n-1 layers of the stack, the volume of the cell nucleus between the n and n-1 layers of the stack Vn can be calculated with the following formula.


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Vn = [Sn + Sn-1 +√

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]

Here h is the scanning step (0.5 μm). The summation of the volumes of the sections gives the total volume of a cell nucleus. For live cell imaging, cells were incubated at 37 °C in a humidified environment with supply of 5% CO2 and 95% air for 24 h. Next, time-lapsed micrographs were captured in an inverted microscope (Leica) with a 10 min interval. EdU Incorporation Assay. EdU (5-ethynyl-2‟-deoxyuridine) is an analogue of thymidine and can be incorporated into DNA strands. Cells that are synthesizing DNA in S-phase of the cell cycle incorporate EdU into the DNA. EdU specific binding fluorescein is introduced to identify cells that undergo DNA synthesis during exposure to EdU.72-77 The EdU incorporation assay was performed using Cell-Light EdU DNA cell proliferation kit (RiboBio, Guangzhou, China), according to the manufacturer‟s instruction. The cells were incubated at 37 oC and 5% CO2 for 48 h. Then, 0.5 μl of 50 μM EdU (RiboBio Co., Ltd.) were added into each well containing 500 μl of the culture medium, and kept for 3 h. The cells were fixed using 4% paraformaldehyde, incubated with 2 mg/ml amino acetic acid for 5 min, and permeabilized with a 0.1% v/v Triton X-100 for another 10 min. In the case of labeling with EdU, the procedure provided with the EdU specific binding fluorescein was followed by adding 100 μl of Apollo 488 solution for 30 min. The samples were subsequently labeled with DAPI to stain the nuclei and observed with an inverted microscope. MTT Assay. The cell viability and proliferation activity on the PLGA micropillars and smooth surfaces were determined using the modified MTT assay.78-83 Briefly, cells were cultured in 12-well TCPs (1 × 104 cells /ml /well) for 6 h, 24 h, 48 h, 72 h and 96 h. Then, 200 l of MTT (Sigma) solution (5 mg/ml) was added into each well. After 4 h of incubation, 700 l of the supernatant in each well was removed, and 1.5 ml of dimethyl sulfoxide (DMSO) was added to dissolve the


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intracellular formazan. The DMSO was also added into the control group in TCPs, after discarding 700 l of the culture medium. In order to detect the absorbance at 490 nm, 300 l of the solution in each well was transferred to 96-well TCPs and measured using a multiskan machine (Thermo Labsystems). DNA Content Determination. The cell proliferation ability was assessed furthermore by determining the total DNA contents of the cells in each well. In the present study, DNA quantification is based on the principle of the method described by Cesarone et al.84 Total DNA contents were determined by the fluorometric analysis on an QM40 fluorescence lifetime spectrometer (PTI, America) using Hoechst 33258 dye (Sigma) according to literature84 with modification. Stock solutions of calf thymus DNA (1 mg/ml) and Hoechst 33258 (200 ng/ml) in sodium phosphate buffer (2 mM Na2EDTA, 0.05 M sodium phosphate, pH 7.0) were prepared. The DNA standards (200-2000 ng/ml) were prepared by adding a given volume of the DNA stock solution to the sodium phosphate buffer. At first, 0.5 ml of the culture medium was replaced with the same volume of the sodium phosphate buffer. All samples and DNA standards were placed in a 37 oC bath for 20 min and then cooled immediately to -80 oC for 30 min. The above process was repeated for 3 times. The cells were destroyed completely to release DNA. Transparent solutions were obtained. One microliter of the Hoechst 33258 dye solution was added into each well. The reaction proceeded for 30 min. The fluorescence emission at 455 nm was measured with the excitation wavelength of 350 nm. DNA content was calculated according to the literature.85 Statistical Analysis. We used One-way analysis of variance (ANOVA) followed by the Fisher LSD test for comparison between two groups. A value of p < 0.05 was considered statistically significant unless otherwise indicated.

RESULTS AND DISCUSSION


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Preparation of Micropillar Array. A PLGA micropillar array was fabricated using a silicon micropit as template. The micropillars were observed with SEM and confocal laser scanning microscopy. Some representative images of an as-prepared micropillar array are shown in Figures 2A and 2B. The images demonstrated quasi-squared micropillars with 3 m width, 6 m spacing and 6 m height. The dimensions of the micropillars were confirmed to be appropriate to induce a severe self deformation of cell nuclei, as shown in Figure 2C about the confocal laser scanning microscopic images of the nuclei of Hela cells on the micropillar array.

Figure 2. Microscopic images of the PLGA micropilllar array and the nuclei of Hela cells on the micropillar array. (A) SEM images of the micropillar array. (B) Confocal laser scanning microscopic image of the micropilllar array. (C) Confocal laser scanning microscopic images of the nuclei of Hela cells on the micropillar array. The distance along the z axis was 4 m. The cell nuclei were stained with DAPI. The fluorescence mode (reflected light imaging) was captured by the standard detector to show cell nuclei (blue), and the transmitted light imaging mode was captured by the transmitted detector to show micropillar arrays (white). The merged image in the


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right of (C) indicated that the nuclei of Hela cells were inserted into the interspace of the micropillars.

Changes of Nuclear Sizes and Shapes of Hela Cells on the Micropillar Array. The cell area and nuclear shape index were calculated based on the normal fluorescence micrographs and confocal laser scanning microscopic images, as presented in Figures 3A and 3B, respectively. The areas of Hela cells were decreased when cultured on the micropillar array compared with that on the smooth surface. A significant decrease of the nuclear shape indexes of Hela cells on the micropillar array are shown in Figure 3C. The average nuclear shape index for Hela cells on the micropillar array was lower than 0.8, indicating a significant cell nuclear deformation.59 The cells possess a tensegrity structure.86-87 External mechanical force is transferred to the cell cytoskeleton by the adhesion molecules when a cell attaches to the extracellular matrix. As a sensor of cell environment topography,88 the cell nucleus is wired to the cytoskeleton by the linker of the nucleocytoskeleton complex, the nuclear pore complex, and the underlying lamina. When the force balance within the cytoskeleton is modified, the nucleus changes its form subsequently.89


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Figure 3. Micrographs and the characterization of the nuclear deformation of Hela cells on the micropillar array and smooth surface. (A) The two dimensional fluorescence micrographs of Hela cells. Prior to observations, the cells were stained to label the nuclei with DAPI, and F-actins with Phalloidin-TRITC. Fluorescence images of the nuclei and microfilaments of Hela cells were taken; red and blue pixels were filtered in converting the grayscale image of the nuclei into green in order to better visualize the image after merged with the corresponding images of the microfilaments shown in red with the green and blue pixels being filtered. (B) 3D confocal laser scanning microscopy of nuclei of Hela cells cultured on the PLGA micropillar array (left) and the corresponding smooth PLGA surface (right). (C) Statistical results of cell area, nuclear shape index, nuclear area, nuclear height and nuclear volume of Hela cells. “***”: p < 0.001 with a significant difference.

The area and height of a cell nucleus were determined according to the confocal laser scanning microscopic images furthermore via 3D microscopy. We carried out


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z-stack 3D scanning of the nuclei of Hela cells cultured on the PLGA micropillar array and smooth surface. The nuclear volumes of Hela cells cultured on the micropillared and smooth surface were calculated according to the formula described in section of MATERIALS AND METHODS in the main text and Figure S1 in Supporting Information. The nuclear volumes of Hela cells were found to be decreased significantly when cultured on the micropillar array for 72 h (Figure 3C). We also found that the projected nuclear area of the Hela cell was decreased while the nuclear height did not change significantly in comparison between in the micropillar array and on the smooth surface (Figure 3C); so the change of the nuclear volume was reflected mainly by that of the projected nuclear area in this study. Proliferation Assays of Hela Cells. Will such a severe nuclear deformation damage the cells? In order to answer the question, the cell proliferation ability was investigated. Firstly, the newly synthesized DNA of the replicating cell was detected by the EdU incorporation assay (Figure 4). Cells growing in the presence of EdU incorporate this compound during S-phase. The fluorophore-labeled azide reacting with the incorporated EdU enabled detection of the green fluorescence by microscopy. The green fluorescence of some nuclei of Hela cells cultured on the PLGA micropillar array for 48 h (Figure 4C) indicated the incorporation of EdU into DNA of cells during replication. The samples were subsequently labeled with DAPI to stain the nuclei. DAPI is bound to the minor groove of double-stranded DNA of cell nuclei. The DAPI-DNA complex has an absorption maximum at wavelength 358 nm (ultraviolet) and its emission maximum is at 461 nm (blue) (Figure 4B). While all of the cells exhibited blue nuclei, some of them during S-phase were positively stained by EdU (Figure 3C), which illustrated that although the cell nuclei were severely deformed on the PLGA micropillar array, the cells could retain their ability to proliferate.


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Figure 4. Principles of EdU cell proliferation assay and DAPI staining, and the fluorescence images of the nuclei of Hela cells on the micropillar array. (A) Principle of EdU incorporation assay. (B) Principle of DAPI staining. (C) Fluorescence images of the nuclei of Hela cells stained with EdU and DAPI on the micropillar array cultured for 48 h. While all of the cells were stained with DAPI (blue), only the cells that were synthesizing DNA incorporated EdU into the DNA strands (green).

We also observed cells on the micropillar in real time, and some live cell images of Hela cells are shown in Figure 5. The cells on the micropillar array rounded up gradually and then divided into two daughter cells in two hours (Figure 5), which was similar to that on the smooth surface (Figure S2).


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Figure 5. Phase contrast micrographs of Hela cells on the PLGA micropillar array. We started to capture images after 24 h of cell seeding, and the starting time was set as 0 min in the pictures. Arrows indicate the cell experiencing division during the record time.

A cell undergoing mitotic proliferation rounds during spindle assembly and then divides via cytokinesis. As shown in Figure 6, the cell migrated on top of the micropillars and rounded up to a near-spherical shape to complete the process of mitosis accompanied by the rounding of the nucleus. Such a process is consistent with the reported mitotic rounding of mammal cells.45, 90 During mitosis, the chromosomes, which have already duplicated, condense and attach to spindle fibers that pull one copy of each chromosome to the opposite side of the cell; the rest of the cell may then continue to divide by cytokinesis to produce two daughter cells, which attach to the substrate subsequently.


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Figure 6. Confocal laser scanning microscopic images of the nuclei of Hela cells on the micropillar array. The cell nuclei (blue) were stained with DAPI. The nucleus turned round which was on top of the micropillars, as indicated by the arrow.

Mitosis is a part of the cell cycle when replicated chromosomes are separated into two new nuclei. During the mitotic phase, including mitosis and cytokinesis, the mother cell is divided into two daughter cells genetically identical to each other. In the process of mitosis, the cells round up to a near-spherical shape to facilitate the organization within the mitotic cell. Mitotic cell rounding limits the space for mitotic spindle to capture, organize and segregate the chromosomes, and ensure spindle pole stability.90-91 A failure to round up may impair the spindle assembly, pole splitting and delay the mitotic progression.90 Thus mitotic cell rounding is essential to cell division. During mitotic rounding, the rounding pressure is increased. The rounding force itself is produced by an osmotic pressure. Osmotic gradients inside and outside the cell induce the water flow and subsequent hydrostatic pressure. The actomyosin cortex


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contraction balances the osmotic pressure in the cell directly. The plasma membrane transporters are considered to regulate the mitotic rounding force. About 10 fold increase in cell cortex tension and hydrostatic pressure is induced during cell mitosis.92 Loss of adhesion is sufficient to induce the isolated cell rounding. External impediments are often encountered for a cell to divide in vivo such as in an epithelium which comprise a densely packed cell layer.45 Actomyosin cortex is required to produce a rounding pressure against the external hindrance for a rounding cell.92 Accumulation of myosin Ⅱ, which requires Cdk1 activation of p21-activated kinase and Rho kinase, is the determinant for the cell to produce persistent cortical tension and rounding force against confinement.93 Precise coordination of the microtubule-based spindle and the actin-based cell cortex guarantees the accurate cell division. Spatial distribution of cortical signals predetermines the spindle orientation and daughter cell positioning.94-95 Under conditions of confinement, such as on the micropillar array, the rounding force pushes the cell out of confinement of the pillars, as shown in Figures 5 and 6, to conduct mitosis unperturbed. Thus the micropillar array mimics the mechanical constraints of the cells in vivo.45 We further employed an MTT assay to quantify activities of cells with deformed nuclei at a series of time to investigate cell proliferation. The MTT assay is a colorimetric assay for assessing cell metabolic activity.79 NAD(P)H-dependent cellular oxidoreductases may, under defined conditions, reflect the number of viable cells.55, 57 These enzymes are capable of reducing the tetrazolium dye MTT to its insoluble formazan, which has a purple color (Figure 7A). Figure S3 confirms a linear relationship between the absorbance at 490 nm and the density of Hela cells.


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Figure 7. Detection of proliferation of Hela cells on the PLGA micropillar array and smooth surface. (A) Principles of the MTT assay. The MTT dye is reduced to formazan by succinate dehydrogenase in a living cell. (B) The absorbance at 490 nm from the MTT assay of cells at the indicated time. (C) Principles of the DNA content detection using Hoechst 33258, which is embedded into DNA. (D) Schematic presentation of excitation and emission of the DNA-Hoechst 33258 complex at the indicated wavelengths, and the statistical data of total DNA content per well determined by fluorometric quantification of the DNA-Hoechst 33258 complex of Hela cells cultured for 72 h. In (B) and (D), n = 4 for each group; a significant difference is indicated by „*‟ (p < 0.05), „**‟ (p < 0.01) or „***‟ (p < 0.001).

The viabilities of Hela cells cultured for 6 h, 24 h, 48 h, 72 h and 96 h are shown in Figure 7B. Although there was no significant difference between the absorbance of cells inocubated on the PLGA micropillar array and smooth surface for 6 h,


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significant decrease of the absorbance was observed afterwards for 24 - 96 h. So, Hela cells exhibited reduced cell proliferation ability on the micropillar array accompanied with severe self deformation of cell nuclei. Total DNA contents of Hela cells seeded on the PLGA micropillar array and smooth surface for 72 h were further determined to assess the cell proliferation ability. The DNA contents were detected according to the emission spectra of DNA-Hoechst 33258. The complex was excited at 350 nm, and the maximum emission wavelength is 455 nm (Figure S4). Figure S5 confirmed a linear relationship between the fluorescent intensity of DNA-Hoechst 33258 and the concentration of DNA. The result in Figure 7D illustrated that the DNA contents of Hela cells seeded on the PLGA micropillar array were lower than that on the smooth PLGA surface. So, it was strengthened that the proliferation of Hela cells was inhibited partially on the micropillar array. Proliferations of HepG2, MC3T3-E1, and NIH3T3 Cells. To further detect the biological responses of the cells with nuclei deformed, the proliferations of MC3T3-E1, HepG2, and NIH3T3 cells were also investigated. Firstly, the newly synthesized DNA of replicating cells seeded on the PLGA micropillar array was detected by the EdU incorporation assay (Figure 8). The green fluorescence of the nuclei of these three cell types on the PLGA micropillar array indicated the incorporation of EdU into DNA and subsequent proliferation of the cells on the micropillar array.


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Figure 8. Fluorescence micrographs of the nuclei of the cells on the micropillar array. Cells that are synthesizing DNA incorporate EdU into DNA strands (green). The cells were stained with DAPI (blue) furthermore.

The nuclear shape indexes, nuclear areas, and cells areas of MC3T3-E1, HepG2, and NIH3T3 cells cultured for 72 h on the micropillar array and smooth surface were detected according to the fluorescence images (Figure 9A). The nuclear shape indexes of all of the cell types were decreased on the micropillar array compared with that on the smooth surface, with the values lower than 0.8 on the micropillars, indicating a significant nuclear deformation.59 Similar to Hela cells, MC3T3-E1 cells exhibited decreased nuclear and cell areas on the micropillar array. In contrast, the nuclear and cell areas of either HepG2 or NIH3T3 cells on the micropillars are similar to those on the smooth surface.


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Figure 9. Proliferation of the indicated cell types (HepG2, MC3T3-E1, NIH3T3) cultured on the micropillar array and smooth surface for 72 h. (A) The nuclear shape index, nuclear areas and cell areas of the indicated cell types. (B) The absorbance value at 490 nm of the indicated cell types from an MTT assay. For each group, n = 4. „*‟: p < 0.05; „**‟: p < 0.01; „***‟: p < 0.001. (C) Total DNA content per well of the cells cultured for 72 h determined by fluorometric quantifications of the indicated cell types. (D) The ratio of proliferation fold as functions of the ratio of the nuclear areas, the ratio of the cell areas, and the ratio of the nuclear shape indexes of the indicated cell types. The proliferation fold is defined as the ratio of the absorbance at 490 nm in an MTT assay for the cells cultured for 72 h to that for the cells cultured for 6 h.

The linear correlations between the absorbance at 490 nm and the cell density of HepG2, MC3T3-E1, and NIH3T3 cells were confirmed, as shown in Figure S6. The results of the MTT assay of the cells cultured for 6 h, 24 h, 48 h, 72 h and 96 h are presented in Figure 9B. Although all of the cell lines exhibited nuclear deformability, there was no significant difference between the absorbance at 490 nm of cells


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incubated on the PLGA micropillar array and smooth surface for 6 h. Significant decrease of the absorbance at 490 nm was shown for the MC3T3-E1 cells cultured for 24-96 h on the PLGA micropillars compared with that on the smooth surfaces, illustrating a reduced cell proliferation on the micropillar array. In contrast, the cell proliferation abilities of HepG2 and NIH3T3 on the PLGA micropillar array did not change with the same absorbance at 490 nm as that on the smooth surface. Total DNA contents of the cells seeded on the PLGA micropillar array and smooth surface for 72 h were further determined to assess the cell proliferation ability. The results are shown in Figure 9C. While the mean DNA content of MC3T3-E1 cells seeded on the PLGA micropillar array was lower than that on the smooth surface, HepG2 cells and NIH3T3 cells on the PLGA micropillar array did not exhibit significantly different proliferation abilities from on the smooth surface. The DNA contents of the cells on the micropillar array and smooth surface are in accordance with the results of the MTT assay. A linear relationship between the absorbance at 490 nm from MTT assay and the DNA content from the fluorescent test as confirmed in Figure S7 for all of the examined cell types including Hela, HepG2, MC3T3-E1, and NIH3T3. To analyze the potential factors that influence the proliferation ability of cells on the micropillar array, the ratio of the proliferation fold was calculated. We used the ratio of the absorbance at 490 nm in the MTT assay for the cells cultured for 72 h to that cultured for 6 h to reflect the proliferation fold. We then tried to establish its relation with some other morphology factors. As shown in Figure S8, we checked the possible relationship between the proliferation fold and nuclear areas, cell areas, and nuclear shape indexes of the cells cultured for 72 h on the micropillar array and the smooth surface. But no correlation was found between them. We then tried more factors, and eventually found that the ratio of proliferation fold was correlated to the ratio of the nuclear areas and the ratio of the cell areas, as shown in Figure 9D. It implies that both nuclear area and cell area may influence cell proliferation. No correlation was detected between the ratio of the proliferation fold


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and the ratio of the nuclear shape index, indicating that the cell proliferation ability on the micropillar array is not significantly affected by the shape change of cell nuclei. Comparison Between All of the Four Cell Types Examined in This Study. We further examined the relations between the nuclear shape indexes and the cell shape indexes. Figure 10 shows the results of all of the four cell types cultured on the micropillar array and smooth surface for 72 h. The cell shape indexes for Hela and HepG2 seemed larger than those of MC3T3-E1 and NIH3T3 cells no matter on the micropillars or on the smooth surfaces. The average nuclear shape indexes and the average cell shape indexes for the four cell types cultured on the micropillars and smooth surface for 72 h are presented in Figure S9; again, Hela and HepG2 cells exhibited larger shape indexes than MC3T3-E1 and NIH3T3. A more severe shape-deformation for MC3T3-E1 and NIH3T3 cells with a more elongated structure is probably related to their fibroblast-like property.61


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Figure 10. The relationship between the nuclear shape indexes and the cell shape indexes. The left and middle rows present the fluorescence images of the indicated cell types cultured on the


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micropillars and the smooth surface for 72 h. Prior to observations, the cells were stained to label the nuclei with DAPI, and F-actins with Phalloidin-TRITC. Fluorescence images of the nuclei and microfilaments of Hela cells were taken in grayscale; then, red and blue pixels were filtered in converting the grayscale image of the nuclei into green, in order to better visualize the image after merged with the corresponding images of the microfilaments shown in red. The shape indexes were calculated according to the formula in the upper left image. For each image, the shape index of the nucleus is marked in the left, and that of the corresponding cell is indicated on the right. The right row of this figure shows the statistical results of shape index of nucleus versus that of the cell for each group. The dashed lines indicate SI = 0.8, below which the quantity SI refers to a significant shape deformation.

The average cell nuclear shape indexes for the examined cell types are all lower than 0.8 (Figure S9), indicating a severe cell nuclear deformation.59 A relatively lower average nuclear shape index was seen for Hela and HepG2 on the micropillar array. The more severe cell nuclear deformability for these two cell types might be due to their epithelial-like property. 61, 96 We found that nuclear shape indexes were not related to cell shape indexes for all the four cell types both on the micropillar array and the smooth surface. Figure 10 and Figure S9 indicated that there is no strict connection between the cell shape and the nuclear shape, although probably the reduction of the cell nuclear size accompanies with the decrease of the cell size. The nuclear shape indexes and nuclear areas of the four cell types cultured for 48 h on the micropillars and smooth surface were also determined. The results are shown in Figure S10A. All cell types exhibited decreased nuclear shape indexes on the micropillars compared with that on the smooth surface. It indicated severe nuclear deformation for all the cell types on the micropillar array. Decreased nuclear areas for Hela and MC3T3-E1 were seen on the micropillars, while no significant difference was found for the nuclear areas of either HepG2 or NIH3T3 cells on the micropillars compared with that on the smooth surface. The ratio of proliferation fold of the cells cultured on the micropillars to that on the smooth surface for 48 h was increased with the ratio of the nuclear areas of the cells on the micropillars to that on the smooth surface (Figure S10B), which is similar to the cells cultured for 72 h. No apparent correlation was shown between the ratio of proliferation fold and the ratio of the


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nuclear shape index of the cells on the micropillars to that on the smooth surface. There was no apparent correlation between the proliferation fold and the average nuclear areas and the nuclear shape indexes for all the cell types cultured on the micropillars and smooth surface (Figure S10C). The fluorescence images of the nuclei of various cells on the micropillar array confirmed the cell type specificity of nuclear deformation (Figures 2C, 3A, 4C, 6, 8). Will such a severe nuclear deformation damage the cells or how about the viability and proliferation ability of cells with nuclei deformed? In order to answer the question, DNA synthesis and cell proliferation of the four cell types on the micropillar array were confirmed firstly by the EdU incorporation assay. We further employed the DNA content determination and the MTT assay to quantify the cell viability and proliferation ability. The proliferation ability of Hela and MC3T3-E1 cells seeded on the PLGA micropillar array was decreased significantly compared with that on the smooth surface. But no influence of the micropillar array on the cell proliferation ability of HepG2 and NIH3T3 cells with the nuclei deformed was observed. From the relationship of the projected nuclear area and nuclear volume of Hela cell, we can see that the nuclear area may reflect the nuclear size in this study (Figure 3C). The decreased cell proliferation ability for Hela and MC3T3-E1 cells cultured on the micropillar array for 72 h was accompanied by the reduced nuclear size (Figure 9). The size of the nucleus has been considered as a reliable indicator of the chromatin condensation.97-98 The nuclear sizes of Hela and MC3T3-E1 cells were decreased significantly on the micropillar array, which indicated that the chromatins of the two cell types were condensed. It seems worthy of indicating another important publication by Guo et al., who have revealed that cell volume change impacts cell stiffness and stem cell fate.99 Our present work about nuclei size and cell proliferation is essentially consistent with their finding of the cell size effect on cell function. Four coordinated processes, including cell growth, DNA replication, chromosome distribution to daughter cells, and cell division constitute the main process of a cell division cycle. In eukaryotes, DNA is synthesized during the S-phase of the cell cycle. DNA synthesis could be altered by chromatin condensation,97 which was, in the


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present study, confirmed by the reduced DNA contents for Hela and HepG2 cells (Figures 7D and 9C) with the decreased cell nuclear size (Figures 9A and S10B) on the micropillars. The quasi-linear relationship between the ratio of proliferation fold and the ratio of the cell nuclear size may indicate that the condensed chromatin decreased DNA synthesis, which finally affects the proliferation of Hela and MC3T3 cells on the micropillars (Figures 9D and S10B). It may interpret why the cell proliferation ability of these cell types was decreased on the micropillar array. We now come to the answers of the questions we addressed in the section of introduction. First, while the cancerous cells derived from the hepatocellular carcinoma (HepG2) examined in this study showed little change of proliferation ability on the micropillar array, similar to the osteosarcoma-derived cells reported by Anselme et al,55 we found that the proliferation ability was obviously decreased for the cancerous cell line derived from the cervical carcinoma (Hela) on the micropillar array of appropriate dimensions with also serious nuclear deformation. Second, despite relatively less significant nuclear SI change of the non-cancerous cell MC3T3-E1, this cell line of mouse preosteoblasts exhibited a significant decrease of proliferation ability of cells on the micropillar array. Another healthy cell line, NIH3T3 (a cell line of mouse fibroblasts), with less significant change of nuclear SI on the micropillar array showed little change of proliferation ability. So, the cell proliferation ability and nuclear shape change have no simple relation with each other, and the change of cellular functions along with the nuclear deformation is highly cell type dependent. Compared to nuclear shapes, nuclear sizes were found to be more relevant to the change of cell proliferation, as summarized in Figure 11.


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Figure 11. The cellular proliferation ability of the four cell types (Hela, HepG2, MC3T3-E1, NIH3T3) on the micropilllar array compared with that on the smooth surface. (A) Process of nuclear deformation and cell proliferation on the micropillar array. The cell undergoing mitotic proliferation migrates onto the top of the micropillars and rounds during spindle assembly and then divides via cytokinesis. (B) Partially inhibited proliferation ability of Hela cells on


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micropillar array compared with that on the smooth surface. (C) Different cell proliferation ability of the indicated cell types on the micropillar array.

Cells respond to surface topography by modifying cell-to-substrate interactions. The nucleus is deformed along with the attachment of the cell plasma membrane to the sides of the pillars. The forces from the stressed interconnected actin fibers were applied onto the nucleus after the establishment of the focal adhesion.55, 100 Nuclei may resist the cytoskeleton tension. The balance between the resistance of a nucleus to the cytoskeleton tension and the stress from the cytoskeleton determines the final nuclear deformation. The cells cultured on the micropillars exhibit fewer actin stress fibers than on flat surfaces.101 Different spreading abilities were observed for different cell types on the smooth surface.102-105 It has been confirmed that cell spreading area is positively correlated with cell adhesion ability.106-108 The highly adhesive ability of Hela and MC3T3-E1cells brings about large spreading areas on the smooth surface (Figures 3C and 9A).102-103 The aggregating property of HepG2 and the highly motile property of NIH3T3 cells imply a lower adhesive ability, leading to small spreading areas on the smooth surface as shown in Figure 9A.104-105 Due to the restriction and guidance of the micropillar array, similar adhesion and spreading behaviors were obtained for all the four cell types on the micropillared substrate. The seriously decreased spreading areas of Hela and MC3T3-E1 cells on the micropillars (Figures 3C and 9A) may reflect smaller nuclear size. As a result, the chromatin was condensed, which led to the partial inhibition of synthesis of DNA and the cell proliferation.64-66, 109

CONCLUSIONS We investigated the proliferation ability of four cell types, Hela, HepG2, MC3T3-E1 and NIH3T3, on the micropillar array. Significant nuclear deformation was found, yet the cells could survive and proliferate on the micropillar array. The change of the cell proliferation ability on the micropillar array compared to on the


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corresponding smooth surface exhibited cell type dependence. The severe nuclear deformation is not harmful for the proliferation of HepG2 and NIH3T3 cells with unchanged nuclear size; in contrast, cell proliferation ability was decreased for Hela and MC3T3-E1 cells with the deformed nuclei accompanied by the reduced nuclear size on the micropillar array. Hence, nuclear deformation of cells on a topographic surface could be reflected not only in change of shape, but also change of size and thus that of chromatin density. Micropillar arrays have the potential to manipulate selective proliferation of the desired cell types in the field of regenerative medicine.

ASSOCIATED CONTENT Supplementary Information Calculation algorithm of the volume of the cell nucleus; The experimentally record mitotic process of Hela cells on the smooth surface; Relationship between the absorbance at 490 nm in an MTT assay and the density of Hela cells; The excitation and emission spectra of DNA-Hoechst 33258; Relationship between the fluorescent intensity of DNA-Hoechst 33258 and the concentration of DNA; Relationship between the absorbance at 490 nm in an MTT assay and the densities of HepG2, MC3T3-E1, and NIH3T3 cells; Relationship between the absorbance at 490 nm from an MTT assay and the DNA contents from the fluorescent tests of Hela, HepG2, MC3T3-E1, and NIH3T3 cells; Relationship between the proliferation fold and nuclear areas, cell areas, and nuclear shape indexes of the cells cultured for 72 h on the micropillar array and the smooth surface; Relationship between the average nuclear shape index and the average cell shape index of the indicated cell types cultured on the micropillar array and the smooth surface for 72 h; Nuclear deformation and cell proliferation for the cells cultured for 48 h on the micropillar array and the smooth surface. The Supporting Information is available free of charge on the ACS Publications website at DOI: ***.

AUTHOR INFORMATION


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Corresponding Author 1

E-mail: [email protected]. Phone: 0086 21 31243506.

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was financially supported by National Natural Science Foundation of China (grants no. 51503043, 51533002, 51703033), and National Key R&D Program of China (grant No. 2016YFC1100300).


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