Critical Areas of Proliferation of Single Cells on Micropatterned

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Biological and Medical Applications of Materials and Interfaces

Critical areas of proliferation of single cells on micropatterned surfaces and corresponding cell type dependence Xiang Yao, Ruili Liu, Xiangyu Liang, and Jiandong Ding ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b03780 • Publication Date (Web): 09 Apr 2019 Downloaded from http://pubs.acs.org on April 13, 2019

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ACS Applied Materials & Interfaces

Critical areas of proliferation of single cells on micropatterned surfaces and corresponding cell type dependence

Xiang Yao, Ruili Liu, Xiangyu Liang, Jiandong Ding*

State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Fudan University, Shanghai 200438, China.

*Correspondence

should be addressed to J.D. DING. E-mail: [email protected]

KEYWORDS: biomaterials; surface patterning; cell-material interactions; cell proliferation; cell type dependence; poly(ethylene glycol) (PEG); arginine-glycineaspartate (RGD)

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ABSTRACT: Material cues to influence cell proliferation are a fundamental issue in the fields of Biomaterials, Cell Biology, Tissue Engineering and Regenerative Medicine. This paper aims to investigate proliferation of single mammal cells on micropatterned material surfaces. To this end, we prepared cell-adhesive circular microislands with 20 areas on the non-fouling background, and systematically examined adhesion and proliferation behaviors of different kinds of single cells (primary stem and non-stem cells, cancer and normal cell lines) on micropatterns. Based on the analysis of experimental data, we found two critical areas about cell proliferation: (1) the critical spreading area of cells from almost no proliferation to confined proliferation, denoted as AP, (2) the critical spreading area of cells from confined proliferation to almost free proliferation, denoted as AFP. We further summarized the relative size relationship between these two critical areas and the characteristic areas about cell adhesion both on patterned and non-patterned surfaces. While proliferation of single primary cells was affected by cell spreading, those cell lines, irrespective of normal and cancer cells, did not exhibit significant cell spreading effects. As a result, this study reveals that proliferation of single cells is dependent upon spreading area, in particular for primary cells on material surfaces.

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1. INTRODUCTION Cell proliferation and corresponding cell type dependence are very important in many cell-pertinent fields. For instance, in vivo structuring processes in the embryonic development, individual growth, and even wound healing are considered to be driven by local differentials of cell proliferation1-6, such as the formation of irregular morphology and even branching structure of tissues and organs. Besides, acquisition of a huge number of seeding cells in tissue engineering or cell transplantation should be based on cell proliferation7-11. Cell proliferation, like many other cell behaviors, is influenced by cell microenvironment, including, albeit not limited to, substrates which cells adhere to. Hence, revealing material cues influencing cell proliferation is a fundamental issue and much important in the fields of Biomaterial, Cell Biology, Tissue Engineering and Regenerative Medicine. Cell spreading effects on cell proliferation has aroused the interest of scientists for decades12-14. For instance, as early as 1978, Judah et al.12 have reported the “cell shape” effect on proliferation of endothelial cells based on a traditional culture system. They used poly(hydroxyethyl methylacrylate) (p-HEMA, a non-fouling polymer) solutions of different concentrations to coat a plastic to adjust cell adhesion, and have provided valuable reference about cell cues on cell proliferation12. Nevertheless, multiple cells were examined in this literature12, and the cell spreading area and shape are always coupled with each other in the traditional culture system. To examine proliferation of a single cell with a relatively confined shape is thus of its own right, which is the main aim of our present publication and is enabled by a surface patterning technique. With the development of advanced material technology, the surface patterning strategy has afforded a powerful tool to control cell adhesion15-20, and intensive studies have made in examinations of cell and material cues on cell adhesion21-27 and differentiation28-39. This kind of technology is also effective for revealing effects of cell cues such as cell spreading and shape on cell proliferation by eliminating other interference factors. By using the powerful micropatterning technique, Chen. et al.21 firstly reported the cell spreading effect on proliferation of primary endothelial cells in -3-



1997, which reveals that cell proliferation was restricted by small spreading. The original description of this literature is “cell growth” instead of cell proliferation characterized by the fraction of DNA synthesis via incubation of 5-bromo-2′deoxyuridine (BrdU). About 15 years later, Versaevell et al.40 mentioned that the proliferation of the primary endothelial cells is also regulated by cell aspect ratio. Besides, a few publications41-42 have confirmed the cell shape effects on the DNA synthesis of primary endothelial and smooth muscle cells on micropatterns, as characterized by the incubation of BrdU within 8-24 hours to estimate the DNA synthesis and hence the fraction of cells in the S phase of cell division. It is so far not clear whether or not a critical spreading area exists about cell proliferation, and how about the cell type dependence of spreading areas effect on cell proliferation. With the development of Cell Biology, Biomaterials and Regenerative Medicine, stem cells have been recognized as a key cell type owing to their multilineage potential, self-renewal ability, and free of ethical problems43-44. Therefore, primary stem and non-stem cells were both evaluated in our study. Herein, in vitro micropatterning culture systems were used to confine spreading of single cells; we choose primary rat mesenchymal stem cells (MSCs), human cervix epithelial carcinoma cells (Hela) and mouse embryo fibroblast cells (NIH3T3) as typical primary, cancer and normal cell lines, respectively. The cell proliferation was examined for as long as 4 days of culture. This paper reports two critical spreading areas about cell proliferation, AP (the characteristic spreading area from nearly no proliferation to confined proliferation for single cells) and AFP (the characteristic spreading area from confined proliferation to almost free proliferation for single cells), as schematically illustrated in Figure 1. Another primary non-stem cell mouse embryo fibroblasts (EF, homologous to NIH3T3 cell) and normal cell line human cervical epithelial cells (HCvEpC, homologous to Hela cell) were also examined to investigate cell type dependent proliferation on adhesive microislands with different areas.

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Figure 1. Schematic presentation of the idea to explore the effects of spreading areas on proliferation of single cells and corresponding critical areas. Ac1, also named A*, the critical area from apoptosis to survival for single cells; AP, the critical area from nearly no proliferation to confined proliferation for single cells; AFP, the critical area from confined proliferation to almost free proliferation for single cells. The horizontal arrow direction here indicates the increase of area of an adhesive microisland on a cellresistant background. In order to persistently confine cell spreading, we developed a micropatterning technique to prepare circular microislands of arginine-glycine-aspartate (RGD) on a non-fouling background. The RGD is a typical oligopeptide and has been widely used for promoting specific cell adhesion45-47. Well-designed gold circular microislands on glass were firstly fabricated by lift-off photolithography, and then cyclic RGD peptides -5-



with thiol end groups were linked onto the gold region via S-Au bonding. PEG is a hydrophilic segment and shows outstanding antifouling properties so as to prevent nonspecific protein adsorption and cell adhesion18, 21. In this paper, a self-assembled monolayer (SAM) of PEG was grafted onto the glass surface (non-gold region) as the non-fouling background. Therefore, different kinds of single cells could be examined in a highly controlled way.

2. MATERIALS AND METHODS 2.1. Fabrication of micropatterns with contrast of cell adhesion. Firstly, circular microislands of gold with 20 kinds of areas (20, 79, 177, 314, 491, 707, 962, 1256, 1590, 1963, 2375, 2826, 3317, 3847, 4416, 5024, 5672, 6359, 7085 and 7850 μm2) or 6 kinds of areas (177, 353, 707, 1413, 2826 and 5652 μm2) on glass were prepared by using a photolithography technique, following our previous protocols18, 48. In brief, glass slides (22 mm  26 mm) were thoroughly washed with piranha solution (concentrated sulfuric acid: hydrogen peroxide = 3:1) for at least 30 min. After ultrasonic cleaning with Milli-Q water and drying with nitrogen gas and oven, a thin layer of photoresist (RZJ-304, Ruihong, Suzhou, China) was applied onto the glass surface by spin-coating (3500 rpm for 20 s), and a pre-designed photomask was covered onto the photoresist surface. After UV light exposure, the samples were developed in a developer solution (RZX-3038, Ruihong, Suzhou, China) for 50 s, and further sputtered with a thin layer of gold on a SBC-12 sputter coater (6 mA for 1.5 min; KYKY, Technology Co., Ltd. China). Then the remained photoresist region (unexposed region) was ultra-sonicated away with acetone for 15 min, and the gold micropatterns on glass were finally fabricated and dried with N2 gas. In order to enhance the cell adhesive contrast of the micropatterned surfaces, we further grafted a SAM of 2-[methoxy(polyethyleneoxy)6-9 propyl] trimethoxysilane (MPEG-Si(OMet)3, tech-90, Gelest) onto the glass surface as an anti-fouling background49. Briefly, the micropatterned glass surface was firstly treated with oxygen plasma for 15 -6-


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min (100 W), and quickly immersed in Milli-Q water for at least 20 min to generate – OH functional groups on the glass (non-gold region). The treated glass was then immersed in about 10 mM M-PEG-Si(OMet)3 in anhydrous toluene with the addition of catalyst (1% triethylamine). The related reaction flask was incubated at 60 ºC oven for 48 h. After PEG grafting, the micropatterned substrates were washed by toluene and ethanol in ultrasonic bath, each for 5 min. After drying with N2 gas, a SAM of RGD ligands was grafted onto the gold microislands to enhance cell adhesion of gold microdomains. Briefly, the micropatterned substrates were immersed in a 25 μM aqueous solution of c(-RGDfK-)thiol (f: D-phenylalanine, K: L-lysine; Peptides International) at 4 ºC overnight. Finally, the micropatterned substrates with contrast of cell adhesion were sterilized by 75% ethanol solution and washed thoroughly with Milli-Q water just before cell seeding. 2.2. Cell culture. Primary rat MSCs were purchased from Allcells Tech., Shanghai; primary mouse embryo fibroblasts (EF) were purchased from Shanghai Gaining Biological Technology Co., Ltd; human cervix epithelial carcinoma cells (Hela) and mouse embryo fibroblast (NIH3T3) cells were purchased from Cell Bank, Type Culture Collection, Chinese Academy of Sciences; human cervical epithelial cells (HCvEpC) cells were purchased from Cell Applications (America). The MSCs were cultured in minimum essential medium α (MEM α, Gibco). EF, Hela, HCvEpC and NIH3T3 cells were cultured in high-glucose Dulbecco’s modified Eagle medium (DMEM, Gibco). All media were with 10% fetal bovine serum (FBS, Gibco) and an additional 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM Lglutamine (Gibco). All cells were cultured in a humidified incubator at 37 C with 5% CO2 atmosphere and passaged at about 80% confluence (digestion with 0.25% trypsinEDTA (Gibco)). As for the primary cells (MSC and EF), only passage 2-3 cells were used for the later experiments. As for the cell lines (Hela, HCvEpC and NIH3T3), “relative passage 2-3” cells after corresponding cell resuscitation were used for the later -7-



experiments. 2.3. Cell seeding and cultivation. After sterilization, the micropatterned substrates of cell-adhesion contrast were transferred into 6 well plates (Corning), and then the above mentioned cells were seeded in each well with 1 × 105 cells unless otherwise indicated. After 1 hour of incubation, the non-adherent cells were removed, and the corresponding 2 mL fresh growth medium was added into each well to culture the cells for another 4 h or 4 d. We also added 0.5 μg/mL aphidicolin (Sigma) into the fresh growth medium to inhibit cell proliferation on the microislands as a control. 2.4. Cell immunofluorescent staining, observation and evaluation. After 5 h (1 + 4 h) or 4 d culture on the micropatterns, all cells were rinsed carefully with phosphate buffer saline (PBS), then fixed with 4% paraformaldehyde for 15 min, and permeabilized with 0.1% v/v Triton X-100 for 5 min before immunofluorescent staining. For nucleus staining, cells were labeled with 2 μg/mL of 4’,6-diamidino-2phenylindole (DAPI, Sigma) for 5 min in order to confirm the cell number on each kind of microislands. As for some filamentous actins (F-actins) staining samples, cells were first incubated with 1 μg/mL of phalloidin-TRITC (Sigma) for 30 min at room temperature before the above mentioned staining of cell nuclei. After rinsed with MilliQ water thoroughly, all the stained samples were observed with an inverted microscope (Axiovert 200, Zeiss) mounted with CCD (AxioCam HRC, Zeiss). To confirm the nuclear volume of single cells (with typical “small”, “middle” and “large” spreading confinement with the microisland areas indicated later in the corresponding results), we captured a series of confocal fluorescence images of cell nuclei (after 5 h culture) with 0.5 μm interval along the thickness of the corresponding nuclei, using a confocal laser scanning microscope (C2+, Nikon, Japan). The nuclear volume was obtained by summation of a stack of serial layers. If Sn and Sn-1 (measured by Image J) 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 approximately -8-


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calculated with the following formula50.

Vn = [Sn + Sn-1 +

𝑆𝑛𝑆𝑛 ― 1 ] × ℎ/3

Here h is the scanning step (0.5 μm). The summation of the section volumes gives the total volume of a cell nucleus. In order to examine the possible cell overlapped growth, we captured a series of confocal fluorescence images of cell nuclei (after 3 d culture) with 0.5 μm interval in the range of the thickness of the nuclei on the corresponding micropatterns, also using the confocal laser scanning microscope (C2+, Nikon, Japan). 2.5. Evaluation of proliferation of single cells. After 5-hour or 4-day culture on the micropatterns, cells were stained by DAPI as above mentioned. Based on the corresponding fluorescence micrographs, we counted the cell number on each kind of microislands. We then calculated the single-celladhesion fraction (FN=1) of each kind of spreading at 5 h (original state) and 4 d (end state), which is defined by the number of microislands with single cell adhesion divided by the number of microislands with single- and multiple-cell adhesions. During cell culture in growth medium, a part of single cells (one cell per microisland) proliferated, hence the number of single-cell-adhesion microislands was decreased, while the total number of single- and multiple-cell-adhesion microislands was kept. Therefore, we define the cell proliferation fraction as the difference of single-cell-adhesion fractions between 5 h and 4 d divided by the fraction of 5 h for each spreading. 2.6. Measurements of the spreading areas of suspended cells. In order to get the average suspending area (Asuspend) of each kind of cells, cells were seeded onto tissue culture plates (TCPS) at the density of 5  104 cells per well in 6well plates. Suspended cells obtained by digestion with 0.25% trypsin-EDTA (Gibco) were seeded onto TCPs, and immediately observed under an inverted microscope (Axiovert 200, Zeiss). Snapshots of suspended cells just falling onto substrate surfaces were captured at the bright field. The project areas of cells were measured by using the -9-



free software Image J. 2.7. Evaluation of DNA synthesis of single MSCs. EdU (5-ethynyl-2’-deoxyuridine) is an analogue of thymidine and can be incorporated into DNA strands instead of thymidine50-58. After culture of MSCs on the RGD micropatterns for 5 h, the EdU (100 μM; Guangzhou RiboBio Co., Ltd., China) contained fresh growth medium was used to replace half of the original growth medium (the working concentration of EdU is 50 μM), and the cells were incubated for another 22 h. Then the cells were fixed with 4% paraformaldehyde for about 20 min and double fluorescently labeled by Apollo® 567 (red, specific binding to EdU and hence indicating the EdU positive cell nuclei) and DAPI (blue, indicating all cell nuclei) according to the manufacturer’s instruction. Stained samples were mounted to glass-bottom plates (Shengyou Biotechnology Co., Ltd, China) for a better vision and observed with an inverted microscope (Axiovert 200, Zeiss) mounted with CCD (AxioCam HRC, Zeiss). The DNA synthesis of single cells with confined spreading was evaluated by the fraction of positive EdU stained single cells on the corresponding microislands. 2.8. Statistic analysis. The statistical data were represented in mean ± standard deviation. Independent experiments joined in statistics, and n = 3 for each group unless otherwise indicated. The one-way analysis of variance (one-way ANOVA) was carried out to evaluate the difference between groups. A difference was regarded as significance when p < 0.05.

3. RESULTS 3.1. Fabrication of micropatterns to achieve single cell adhesion. After combination of lift-off photolithography and SAM techniques, we successfully fabricated a series of circular RGD microislands with different areas on the non-fouling PEG background. Typical micropatterns are presented in Figure 2A.

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Figure 2. Initial adhesion of MSCs on the designed microislands with varied areas. (A) Bright field micrograph of as-fabricated micropatterns of RGD-grafted gold microislands and schematic presentation of the side view of micropatterns. The selfassembled monolayer of PEG here affords a non-fouling background, and the RGD monolayer on the Au microislands affords cell-adhesive microdomains. We generated microislands with 20 diameters including 5, 10, 15, 20, 25, 30, 35, 40, 45 μm (line 3), 50, 55, 60, 65, 70, 75, 80 μm (line 2), 85, 90, 95, 100 μm (line 1). (B) Phase contrast micrograph of the initial MSCs (5 h after cell seeding) on the microislands with varied areas. (C) Fluorescence micrograph of nuclei of MSCs corresponding to Figure 2B to show the cell number on each kind of microislands. (D) Confocal fluorescence micrographs of single MSCs (5 h after cell seeding) on the typical microislands with varied areas. Red: F-actins, blue: nuclei. The dashed circles here indicate the contour of the underlying RGD-grafted microislands. Some phase contrast and fluorescence micrographs of MSCs on the fabricated micropatterns after 5 hours of culture are shown in Figures 2B-C. The cells were well localized by the designed adhesive microislands on the non-fouling background. Fluorescence images of F-actins and nuclei of some typical single MSCs on the microislands with varied areas are presented in Figure 2D. The cell size and shape were - 11 -



well controlled by those of pre-designed microislands based on our surface patterning technique. 3.2. Critical areas for cell adhesion on micropatterned surfaces. According to the initial cell adhesion on the 20 kinds of circular microislands, we confirmed two characteristic areas about MSC adhesion, as schematically presented in the upper row of Figure 3. The critical adhesive area for a single cell from apoptosis to survival is denoted as Ac1 (or A*); the critical area of a microisland from single-cell adhesion to multi-cell adhesion is defined as Ac2.

Figure 3. Fraction of cellular occupation per microisland and corresponding characteristic areas for MSC adhesion. The upper row schematically shows two characteristic areas about cell adhesion, Ac1, the critical adhesion area for a single cell from apoptosis to survival, and Ac2, the characteristic area of a microisland from singlecell adhesion to multiple-cell adhesion. The arrow direction here indicates the increase of area of an adhesive microisland on a cell-resistant background. The lower row shows the statistical results of fN ＞ 0 (the number of single- and multiple-cell-adhesion microislands divided by the total number of microislands) and fN ＞ 1 (the number of multiple-cell-adhesion microislands divided by the total number of microislands) per microisland as a function of adhesive area of microislands after 5-hour culture. The characteristic areas Ac1 and Ac2 were obtained from the crosspoints between the corresponding marked asymptote lines.

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In this study, a microisland with either one cell or multiple cells is regarded as “occupied”. The occupation fraction fN>0 (N indicating cell number) is defined as the number of single- and multiple-cell-adhesion microislands divided by the number of the total statistical microislands. fN>0 (red dots) of MSCs as a function of microisland area (adhesive area) is shown in the lower row of Figure 3. The probability of cells to adhere on a microisland was zero on the sufficiently small microisland. As the adhesive area increase, fN>0 gradually increased from zero to unity, and demonstrated a transition from “non-occupied” to “occupied” microisland, implying a switch from cell “apoptosis” to “survival”. The crosspoint of two tangential lines was employed to determine the critical transition point, as shown in Figure 3. In this way, we obtained the characteristic area from apoptosis to survival Ac1, similar to the determination of critical micelle concentration (CMC) from optical density (OD) as a function of solute concentration in studies of the micellization transition of amphiphiles in a selective solvent59-60. The Ac1 for MSCs is about 100 μm2 according to Figure 3. We define a microisland with at least two cells adhered as “multi-occupied”. The multi-occupation fraction fN>1 is calculated from the number of multiple-cell-adhesion microislands divided by the total statistical number of microislands, and the corresponding data of MSCs with varying areas of microislands are shown in the lower row of Figure 3 (blue dots). The curve of fN>1 versus microisland area (adhesive area) also exhibited a CMC-like transition. Therefore, the characteristic area from singlecell-adhesion to multiple-cell-adhesion Ac2 could be determined as the crosspoint between the corresponding marked blue asymptote lines. The Ac2 for MSCs is about 500 μm2 according to Figure 3. According to the bright field micrograph of suspended MSCs just falling onto TCPs, as presented in Figure S1, we measured the averaged project area of suspended cells, which is denoted as Asuspend, for the convenience of comparison of characteristic areas of cells on patterned and non-patterned surfaces. The Asuspend for MSCs is about 300 μm2 according to our statistical results. - 13 -



Besides, we also measured the three characteristic areas (Ac1, Ac2 and Asuspend) of EF, Hela and NIH3T3 cells in the same way, with results presented in Table S1 and Figures S2-S3. All these results illustrated that different kinds of cells presented different characteristic areas about cell adhesion but with similar trends: Ac1 < Asuspend < Ac2. 3.3. Critical areas for cell proliferation on micropatterned surfaces. After a few days of culture, parts of single cells on the micropatterns proliferated, and the corresponding occupied microislands changed from “single” (single-occupied) to “multiple” (multi-occupied), as schematically presented in Figure 4A. As the total number of single- and multi-occupied microislands might be kept constant during cell culture, the fraction of single cells FN=1, which is calculated from the number of singleoccupied microislands divided by the total number of single- and multi-occupied microislands, must decrease with cell proliferation. So we firstly obtained the FN=1 of the initial state (5-hour culture) and final state (4-day culture) and then calculated the proliferation fraction of single cells (P) with different kinds of confined areas (microisland area) according to the corresponding change of FN=1. The statistical results of FN=1 and the calculation formula of P are illustrated in Figures 4B and 4C. In order to investigate the effectiveness of our statistical method about proliferation of single cells, we added a proper concentration (0.5 μg/mL) of the cell proliferation inhibitor aphidicolin32-33 into the culture system and compared the proliferation of single cells (6 kinds of spreading areas, the corresponding micropatterned substrate presented in Figure S4) with or without aphidicolin, as shown in Figure S5. The statistical results illustrate that the proliferation fraction of single cells with addition of the cell proliferation inhibitor (“4 days + Aphi”) is much smaller than that of control group (“4 days”) in each kind of spreading, as clearly shown in Figure S5C. This methodology correctively evaluated the proliferation inhibition ability of aphidicolin and hence indirectly proved the effectiveness of our statistical approach to evaluate proliferation of single cells.

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Figure 4. Proliferation behavior of single MSCs with varied spreading areas after 4 days of culture. (A) Schematic presentation of proliferation of single cells with varied confined spreading. The single-cell-adhesion microislands become multiple-celladhesion microislands as cells proliferate, while the total number of single- and multiple-cell-adhesion microislands is a constant. (B) Statistical results of single cells fraction (FN=1, the number of single-cell-adhesion microislands divided by the total - 15 -



number of single- and multiple-cell-adhesion microislands) after 5-hour and 4-day culture. The confined spreading areas are 20-7850 μm2. For the smallest group (about 20 μm2), cells were barely observed on the microislands, so we designated the values of single cell fraction of this group are 100% both after 5-hour and 4-day culture. (C) Schematic presentation of the calculation of proliferation fraction of single cells (P, during the period of t1 to t2) from the date of single cell fraction (FN=1). (D) Statistical results of the proliferation fraction of single cells per microisland as a function of adhesive area of microislands (logarithmic coordinates). The critical area AP is defined as the crosspoints between the corresponding two marked asymptote lines. (E) Statistical results of the proliferation fraction of single cells per microisland as a function of adhesive area of microislands. The characteristic area AFP is defined as the crosspoints between the corresponding two marked asymptote lines. (F) Schematic presentation of the relative size relationship of different kinds of characteristic areas about MSC proliferation (red tag) and adhesion (blue tag). After 4 days of culture, the calculated proliferation fraction of single MSCs on 20 kinds of microislands (area: 20-7850 μm2) are shown in Figure 4D (logarithmic coordinates). The proliferation of primary MSCs was obviously affected by cell spreading. The proliferation fraction of single cells was almost zero on the sufficiently small microislands. As the confining area (microisland area) increased, the proliferation fraction of single cells gradually increased from zero to unity, and demonstrated a transition from “no proliferation” to “proliferation”. The crosspoint of two tangential lines was employed to determine this critical point in Figure 4D. In this way, we obtained the critical area from nearly no proliferation to confined proliferation AP for the first time, also similar to the determination of CMC of amphiphiles in a selective solvent59-60. The AP for MSCs is about 300 μm2 according to Figure 4D. Similarly, the characteristic area from confined proliferation to almost free proliferation AFP could be determined as the crosspoints between the corresponding marked asymptote lines in Figure 4E. The AFP for MSCs is about 1900 μm2 according to Figure 4E. We further compared all of the above mentioned characteristic areas about MSC adhesion and proliferation. The relative size relationships are schematically presented in Figure 4F, which illustrates that the AP is larger than Ac1, but smaller than Ac2, and almost equally to Asuspend. Besides, the AFP is much larger than AP and Ac2 for primary - 16 -


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MSCs. 3.4. DNA synthesis and nuclear features of single MSCs with varied spreading areas. The above-mentioned results have proved that cell proliferation is significantly regulated by cell spreading. But why does it happen? We then did some preliminary research to illustrate the corresponding reasons by using a few typical circular microislands (177-5652 μm2, as shown in Figure S4). EdU is a kind of thymidine analogues, and can compete with thymidine to bind to DNA strands during DNA synthesis50-51, 54-56. Therefore, the DNA-synthesizing nuclei with EdU incubation are stained positively, and otherwise nuclei are stained negatively, as shown in Figure 5A. The DNA synthesis of single MSCs with confined spreading areas were evaluated by the percentage of EdU positively labeled single cells (and hence the percent of cells in the S phase of cell division), as presented in Figure 5B. The DNA synthesis fraction of single cells decreased with the decrease of cell spreading. This size dependence of DNA synthesis of primary MSCs is in accordance with our above-mentioned statistics of MSC proliferation.

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Figure 5. DNA synthesis of single MSCs with confined spreading areas. (A) Phase contrast and fluorescence micrographs of single MSCs on the typical microislands after 22-hour incubation of the EdU contained culture medium. Upper row shows a typical EdU positive stained sample. Lower row shows a typical EdU negative stained sample. All cell nuclei were labeled by DAPI (blue) and EdU contained nuclei were labeled by Apollo567 (red). The dashed circles indicate the contours of the underlying RGDgrafted microislands, and the solid lines indicate the corresponding cell contours on the microislands. (B) Statistical results of the fraction of EdU positive labeled single MSCs, which reflects the DNA synthesis behaviors of single MSCs with confined spreading areas. The p values of one-way ANOVA of the data in B are listed in Supplementary Table S2. We further used a series of confocal microscopic images of nuclei of single cells (as schematically illustrated in Figure 6A) to measure the nuclear volume of single cells with typical “small”, “middle” and “large” spreading areas. Statistic results are shown in Figure 6C, while the detailed distribution diagram of large and small conditions are presented in Figure 6B. Although the average nuclear height of single cells is little higher (as shown in Figure 6D) on small microislands than on large microislands, the nuclear volume is much smaller on small microislands, which indicates nucleus - 18 -


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condensation on small microislands, just as the similar trend in some other reports40-42. Besides, we also measured the relative fluorescence intensity and project area of nuclei of single MSCs with varied spreading areas, as shown in Figures S6 and S7 respectively. The relative nuclear fluorescence intensity (related to the chromatin density) increased with decrease of cell spreading area, and the project area decreased seriously with the decrease of cell spreading area, which also indirectly indicates the chromatin and nucleus condensation to some extent on the small or restricted microislands.

Figure 6. Nuclear volume and height of single MSCs with confined spreading areas after 5 hours of culture. (A) Schematic presentation of the acquisition of confocal micrographs of cell nuclei. (B) The distribution diagram of nuclear volume of single MSCs on the relative large (2826 μm2) and small (177 μm2) microislands. (C) Statistical results of nuclear volume (mean ± SD) of single MSCs with typical confined spreading areas after 5-hour culture. (D) Statistical results of nuclear height (mean ± SD) of single MSCs with typical confined spreading areas after 5-hour culture. Statistical results come from the confocal micrograph of nuclei of single MSCs (n ≥ 30 for each spreading) labeled by DAPI. The p values of one-way ANOVA of the data in C and D are listed in Supplementary Tables S3 and S4. Based on these experiment results, we speculated that the chromatin and nucleus - 19 -



condensation on the small microislands is probably the main reason for the restricted DNA synthesis and hence the lower fraction of cell proliferation for cells with small spreading. This is consistent with the speculation about some primary non-stem cells reported in the literature40-42. 3.5. Proliferation behavior of other primary cells. Through careful experimental design and statistics, we suggested and confirmed two characteristic areas (AP and AFP) about proliferation of primary stem cells and their relative size relationship with some characteristic areas about cell survival and adhesion, such as Ac1, Ac2 and Asuspend, as schematically presented in Figure 4F. Do other primary cells exhibit similar characteristic areas? In order to answer this question, we chose another primary non-stem cell EF as an example to evaluate their proliferation behavior with 20 kinds of confined spreading areas. The statistical results of EF cells with varied spreading areas are presented in Figure 7. By using the same method as MSCs, we also obtained these two characteristic areas (AP and AFP). The AP for EF is about 400 μm2 according to Figure 7B and the AFP is about 2000 μm2 according to Figure 7C. Similarly, we measured Ac1, Ac2 and Asuspend for EF, as shown in Table S1 and Figure S2. The size relationship about those characteristic areas is summarized and schematically presented in Figure 7D. Combined with Figure 4F, both primary cells exhibited critical areas about cell proliferation and presented similar relative size relationship: the AP is larger than Ac1, but smaller than Ac2, and almost equally to Asuspend; the AFP is much larger than AP and Ac2 for the corresponding cells.

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Figure 7. Proliferation behavior of single EF cells with varied confined spreading areas after 4 days of culture. (A) Statistical fractions of single EF cells after 5-hour and 4-day culture. The confined spreading areas are 20-7850 μm2. (B) Statistical results of the proliferation fraction of single cells per microisland as a function of adhesive area of microislands (logarithmic coordinates). The critical area AP is defined as the crosspoint between the corresponding two marked asymptote lines. (C) Statistical results of the proliferation fraction of single cells per microisland as a function of adhesive area of microislands. The characteristic area AFP is defined as the crosspoint between the corresponding two marked asymptote lines. (D) Schematic presentation of the relative size relationship of different kinds of characteristic areas of EF cells. 3.6. Proliferation behavior of cell lines on micropatterned surfaces. We further examined the effects of confined spreading on proliferation of cell lines, and something much different was found. We chose a typical cancer cell line (Hela) and normal cell line (NIH3T3) as examples to investigate the proliferation behavior of single cells with varied confined spreading areas. Note that NIH3T3 is homologous to our previously studied primary EF cells in order to exclude the influence between - 21 -



different cell sources in comparison of proliferation of cell lines and that of primary cells. Proliferation of single Hela cells with confined spreading areas after 4 days of culture is shown in Figure 8A, which presents a big difference with above-mentioned primary cells (Figures 4 and 7). There is an obvious mutation process near the area of Ac1: when cell spreading smaller than Ac1, the single cells were of apoptosis and hence the proliferation fraction was almost zero, while a high portion (about 80%) of single Hela cells proliferated to multiple cells when cell spreading was larger than Ac1. Based on this proliferation phenomenon and the definition of AP and AFP, the AP of Hela is almost equal to their Ac1, and the AFP of Hela is near their AP but smaller than their Asuspend. Those two characteristic areas about cell proliferation are both different from those of the above mentioned primary cells. Proliferation behavior of single NIH3T3 cells with confined spreading areas after 4 days of culture are shown in Figure 8B. The typical normal cell line shows a similar proliferation behavior to that of the cancer cell line. Therefore, the AP of NIH3T3 is close to their Ac1, and the AFP of NIH3T3 is near their AP but smaller than their Asuspend according to the definition of critical areas about cell proliferation. The similar relative size relationship of characteristic areas of typical cancer and normal cell lines are schematically illustrated in Figure 8C, which shows the difference with primary cells more intuitively (Figure 8C vs Figure 4F).

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Figure 8. Proliferation behaviors of single Hela and NIH3T3 cells with confined spreading areas after 4 days of culture. (A) The upper row shows the statistical results of fraction of single Hela cells. The lower row shows the statistical results of the proliferation fraction of single Hela cells with confined spreading areas. The crosspoints of dashed line and x-axis indicate the Ac1 and Asuspend of Hela cells respectively. (B) The statistical results of NIH3T3 cells. (C) Schematic presentation of the relative size relationship of different kinds of characteristic areas of Hela and NIH3T3 cells. We further chose a special normal cell line HCvEpC, which is homologous to Hela cells, in order to exclude the influence between different cell sources when we compared a cancer cell line and a normal cell line. The proliferation behavior of single - 23 -



Hela and HCvEpC cells with six kinds of typical confined spreading areas after 4-day culture are shown in Figure S8. Again, the normal cell line shows a similar proliferation behavior of the corresponding cancer cell line on confined micropatterns: even as smaller as the Asuspend of corresponding cells, a large portion (about 90%) of single HCvEpC cells can proliferate to multiple cells, just the same as Hela cells. This result strengthens the similar proliferation behavior between normal and cancer cells on micropatterns if both of them are from cell lines instead of primary cells. Through careful statistics, we also found decreased nuclear spreading on the small microislands for the Hela and NIH3T3 cells, as shown in Figure S9, which again indirectly hinted nucleus and chromatin condensation on small microislands. However, proliferation of the cell lines is not significantly restricted by small cell spreading. Combined with the reported proliferation of cancer cells61-68, we speculated that as the cell lines passaged over and over again in vitro, the tested normal cell lines may have a characteristic of cancer cells, namely, the ability of an overlapped growth. Therefore, we further used a confocal microscope to observe the 3D reconstructed structure of cell nuclei on typical micropatterns, as presented in Figures 9 and S10. The nuclear section views clearly illustrated that compared to the primary cells (MSC and EF), the normal cell lines (both NIH3T3 and HCvEpC) are easier to form an overlapped growth on confined geometry (similar to Hela cells).

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Figure 9. Typical 3D reconstructed confocal micrographs and corresponding section views of nuclei of indicated cells (labeled by DAPI) on the confined microislands after 3 days of culture. The dashed circles indicate the contour of the underlying RGDgrafted microislands. The horizontal solid lines here indicate the corresponding positions of section view.

4. DISCUSSION This study addresses a question whether or not there are critical areas about cell proliferation quantitatively. In order to answer this question, we designed and fabricated a series of adhesive microislands with 20 kinds of areas on a non-fouling background. Different microislands on the same microscale region rule out the interference of paracrine soluble factors and substrate properties, which otherwise must happen if one - 25 -



changed just average spreading areas of cells simply by using different cell densities on a non-patterned surface. Before the investigation of critical areas about cell proliferation, we first examined three characteristic areas (Ac1, Ac2 and Asuspend) about cell adhesion, which may be related to cell proliferation. For the primary MSCs (stem cell), the Ac1 is about 100 μm2, the Ac2 is about 500 μm2 and the Asuspend is about 300 μm2. We also measured the Ac1, Ac2 and Asuspend of primary non-stem cell (EF), normal cell line (NIH3T3) and cancer cell line (Hela) (Table S1 and Figures S2-S3) in the similar way. All these results demonstrated that different kinds of cells presented different characteristic areas about cell adhesion but exhibited similar trends: Ac1 < Asuspend < Ac2. The relative size relationship of characteristic areas about cell adhesion is universal according to our present studies, no matter for the primary cells or cell lines. The size dependence of cell adhesion and that relationship of characteristic areas about cell adhesion are consistent with previous reports21, 24. Then we focused on the investigation of critical areas about cell proliferation. The proliferation of primary MSCs was obviously affected by cell spreading, as clearly illustrated in Figure 4. As the microisland area increased, the proliferation fraction of single cells increased from zero to unity, and demonstrated a transition from “no proliferation” to “proliferation”. We then determined the critical area from almost no proliferation to confined proliferation AP (about 300 μm2). Similarly, the characteristic area from confined proliferation to almost free proliferation AFP (about 1900 μm2) were also obtained. The above study illustrated the existence of critical areas about cell proliferation at single cell levels and confirmed the restriction effect of small spreading on primary cell proliferation. We further compared all of the above mentioned characteristic areas about cell adhesion and proliferation. The relative size relationships (Figure 4F) clearly illustrated that the AP is larger than Ac1, but smaller than Ac2, and almost equal to Asuspend for MSCs. Besides, the AFP is much larger than AP and Ac2.

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In order to interpret why the proliferation of MSCs was affected by cell spreading, we further prepared six kinds of typical circular microislands to evaluate the DNA synthesis and nuclear features of single cells on adhesive microislands. The results (Figures 5-6) illustrate that the fraction of DNA synthesis of single cells is decreased with the decrease of cell spreading area, so is the nuclear volume of single cells. This size dependence of DNA synthesis and nuclear volume is in accordance with publications about primary non-stem cells (endothelial and smooth muscle cells)21, 4042.

Besides, the increased fluorescence intensity (Figure S6) of cell nuclei on small

microislands also indicated chromatin condensation with less cell spreading. According to these results and pertinent references40-42, we speculated that the chromatin and nucleus condensation on the small microislands might account for restricted DNA synthesis and hence lower fraction of proliferation for single cells with less spreading. We then examined another primary EF cells (non-stem cell), the relative size relationship of critical areas about cell proliferation and adhesion shows a similar trend to that for primary MSCs (Figure 7D vs Figure 4F). These results further confirmed the spreading effect on primary cell proliferation. Besides, the proliferation fraction of EF on large microislands (Figure 7C vs Figure 4E) is much smaller than MSCs, which confirmed a relative stronger self-renewal ability of primary stem cells. In short, by using our developed surface micropatterning technique, we have, for the first time, revealed the existence of critical areas AP and AFP about cell proliferation; their relative size relationship with the characteristic areas of cell adhesion obey a similar trend in both primary stem and non-stem cells. We further examined cell lines. Typical cancer cell line Hela, normal cell lines NIH3T3 (homologous to EF) and HCvEpC (homologous to Hela) were chosen as examples in order to exclude the influence between different cell sources. Hela cells presented a different dependence of proliferation on spreading area from the primary cells (Figure 8 vs Figure 4). Nearby the corresponding Ac1 of Hela cells, a distinct mutation of proliferation fraction change was found. Based on the definition of AP and AFP, we can inform that the AFP of Hela cells is almost equal to their AP, and both critical - 27 -



areas are close to their Ac1, while smaller than their Asuspend. Those two critical areas about cell proliferation are both quite different from those for primary cells. It is very interesting that the typical normal cell line (NIH3T3) shows a similar proliferation behavior to cancer cell line (Hela), yet quite different from the homologous primary EF cells. Another comparison between HCvEpC and its homologous Hela cell (Figure S8) further confirmed the similar proliferation behavior between normal and cancer cells on micropatterns, if both of them are cell lines. Unlike primary cells, why is proliferation of cell lines restricted by small cell spreading very insignificantly? For cancer cells, this can be easily understood from overlapped cell growth and infinite proliferation of cancer cells61-68; but for normal cell lines, it is a bit unexpected. We speculated that as the cell lines passage over and over in vitro, the tested normal cell lines may have some characteristics of cancer cells. Then, we further used our confocal microscope to observe the 3D reconstructed structure of cell nuclei on typical micropatterns. Figures 9 and S10 clearly illustrated that compared to the primary cells (MSC and EF), the normal cell lines (both NIH3T3 and HCvEpC) form an overlapped growth more easily, just similar to cancer cell line (Hela) on micropatterns. Recently, Roy et al69 have also reported an overlapped growth of NIH3T3 cells on a micropatterned surface when they examined cell cluster behaviors without exogenous biochemical factors; and they further investigated nuclear reprogramming of those laterally confined cells. Hence, it is reasonable that the overlapped growth happened on the confined micropatterns for both the examined normal and cancer cell lines, which weakened the restriction of cell spreading on cell proliferation on the 2D micropatterned surface, as schematically summarized in the right of Figure 10. Therefore, a mutation change process of the proliferation faction could be seen near Ac1, and the AP almost equaled their Ac1. Besides, the AFP is relatively closer to AP but smaller than their Asuspend for all of the cell lines. As for the primary cells, MSC and EF cells are more difficult to form an overlapped growth on micropatterns. The nucleus and chromatin condensation of single cells on small microislands probably restrict DNA synthesis and finally confined - 28 -


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cell proliferation, as schematically summarized in the left part of Figure 10. So, a relatively slow growth process of proliferation fraction under small spreading conditions could be seen, and the AFP is much larger than AP and Asuspend for both of the primary cells, while quite different from cell lines.

Figure 10. Schematic presentation of the cell spreading and cell type dependent cell proliferation examined in this study.

5. CONCLUSIONS By using our developed micropatterning technique, we examined proliferation of single cells (primary cells and cell lines) on adhesive microislands of a series of areas. Both primary stem cells and non-stem cells proliferated in a manner significantly - 29 -



affected by cell spreading areas. We put forward, for the first time, the terms of two critical spreading areas AP and AFP about cell proliferation, one is from almost no proliferation to confined proliferation, and the other is from confined proliferation to almost free proliferation. All of the examined primary cell types show similar relative size relationships of characteristic areas. A further study implied that the chromatin condensation on the small spreading condition might be the main reason of the lower level of DNA synthesis and then restriction of cell proliferation. Unlike primary cells, the normal cell lines (HCvEpC and NIH3T3 cells) present similar behaviors to the cancer cell line (Hela cells) with weakened restriction of small spreading on cell proliferation. The spreading effect on cell proliferation and corresponding cell type dependence shed new insight into basic cell behaviors, and our methodology to determine critical areas of single cells could be extended to study adhesion and proliferation of other cells. The concept of critical areas of proliferation of single cells on micropatterns is helpful for cell research on various material surfaces.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: ***. Bright field micrograph of suspended MSCs just falling onto tissue culture plates; Statistical results of the characteristic areas about cell adhesion; Fraction of cellular occupation per microisland and corresponding characteristic areas for adhesion of EF cells; Fraction of cellular occupation per microisland and corresponding characteristic areas for adhesion of Hela and NIH3T3 cells; Bright field micrograph of as-fabricated micropatterns of RGD-grafted gold microislands; Proliferation behavior of single MSCs with confined spreading areas after 4-day culture with or without the addition of aphidicolin; The p values of one-way ANOVA of the data in Figure 5B for DNA synthesis of single MSCs on microislands with different spreading areas; Distribution - 30 -


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diagram of relative fluorescence intensity of nuclei of single MSCs with confined cell spreading areas; Distribution diagram of project area of nuclei of single MSCs; The p values of one-way ANOVA of the data in Figure 6C for nuclear volume of single MSCs on microislands with different spreading area; The p values of one-way ANOVA of the data in Figure 6D for nuclear height of single MSCs on microislands; Proliferation behavior of single Hela and HCvEpC cells with confined spreading; Distribution diagram of project area of nuclei of single Hela and NIH3T3 cells with confined cell spreading areas; 3D reconstructed confocal fluorescence micrographs of cell nuclei on the small microislands.

AUTHOR INFORMATION Corresponding Author *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 (grant no. 51703033 and 51533002), National Key R&D Program of China (grant No. 2016YFC1100300), Science and Technology Commission of Shanghai Municipality (grant No. 17JC1400200), and China Postdoctoral Science Foundation (grant no. 2017M621358).

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Sci. U. S. A. 2018, 115, E4741-E4750.

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Graphic for Table of Contents

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Critical Areas of Proliferation of Single Cells on Micropatterned

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