Three-Dimensional Microstructured Poly(vinyl alcohol) Hydrogel

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Three-Dimensional Microstructured Poly(vinyl alcohol) Hydrogel Platform for the Controlled Formation of Multicellular Cell Spheroids Xiao-Qiu Dou, Ping Li, and Holger Schönherr Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01345 • Publication Date (Web): 21 Nov 2017 Downloaded from http://pubs.acs.org on November 25, 2017

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Three-Dimensional

Microstructured

Poly(vinyl

alcohol) Hydrogel Platform for the Controlled Formation of Multicellular Cell Spheroids Xiaoqiu Dou,‡* Ping Li,‡ and Holger Schönherr* AUTHOR ADDRESS: Physical Chemistry I and Research Center of Micro and Nanochemistry and Engineering (Cµ), Department of Chemistry and Biology, University of Siegen, AdolfReichwein-Str. 2, 57076, Siegen, Germany KEYWORDS: Muticellular Spheroids, Three-Dimensional, Hydrogel, Mirowells, Drug Testing

ABSTRACT: Three-dimensional (3D) multicellular cell spheroids (MCSs) are excellent in vitro cell models, in which e.g. the in vivo cell-cell interaction processes are much better mimicked than in conventional two-dimensional (2D) cell layers. However, the difficulties in the generation of well-defined MCSs with controlled size severely limit their application. Herein, low-adhesive poly(vinyl alcohol) (PVA) hydrogels structured with inverted pyramid-shaped microwells were used to guide the aggregation of cells into MCSs. The cells settling down into the microwells by gravity accumulate at the central tip of the wells and then gradually grow into spheroids. The size of cell spheroids can be straightforwardly controlled by the culture time and initially seeded cell number. The MCSs generated in a parallel microarray format were further used for drug testing. Our results suggest in agreement with complementary literature data that

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the cell culture format plays a critical role in the cellular response to drugs, and also confirms that spheroids possess a much higher drug resistance than cells in 2D layers. This novel microstructured PVA hydrogel is expected to offer a potential platform for the facile preparation of spheroids for various applications in the biomedical field.

INTRODUCTION During many biological processes, cell-cell interactions, contacts and communications are important for maintaining cellular function and for regulating proper signaling pathways. As a consequence, when cells are isolated from native tissue and are subsequently cultured in vitro, the ability to reintroduce such cellular contacts can be critical for preserving cellular viability and phenotype. The ability to successfully do so may lead to advances in diverse fields, from cell biology to the medical field.1 Compared to traditional two-dimensional (2D) cell culture, which may lead depending on cell type and culture conditions to spread-out cells in sub-monolayers or to confluent monolayers, multicellular spheroids (MCSs) very frequently better resemble the physiological environment.2 This has been increasingly studied in the context of tissue, organ, and tumor models because of enhanced cell-cell contacts and cellular function.3 For example, cancer-related cells cultured into MCSs mimicking a tumor microenvironment have been used to study metastasis, cell migration, or have been even used in drug screening. 4 To mimic early embryogenesis and to improve cell survival and differentiation, embryonic stem cells are routinely cultured into spheroids known as “embryoid bodies”.5 In addition, neuronal precursor cells are aggregated into neurospheres to achieve their multipotency and proliferation potentials.6 Finally, mesenchymal stem cells cultured into spheroids can promote anti-inflammatory, angiogenic, and tissue reparative effects.7


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Based on the merits of MCSs, a variety of techniques has been explored to promote the formation of uniform-sized cell spheroids, including the use of micro-textured surfaces,8 hanging drop technique, 9 chemical micropatterns, 10 thermoresponsive polymers, 11 cell encapsulation in hydrogels

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or rotary bioreactors,

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and so on.

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Recently, taking advantages of

microfabrication advancements, designed microstructures have drawn a lot of attention in the field of in vitro cell culture to form high-quality cell spheroids in a more controlled fashion.16 A more widely exploited microstructure format is the microwell format, which provides a confined environment for cell growth, enhancing cell-cell interactions and consequently promoting the formation of MCSs within the microwells. 17 , 18 The preparation of cell spheroids using this method does not require any special bioreactor and is thus compatible with traditional 2D cell culture. Moreover, the size of the generated cell spheroids in microwells is spatially and temporally controllable. However, most of the reported microwells possess flat or round bottoms, which may allow entrapped cells to spread on the bottom surface and hence results in 2D cell growth behavior instead of the desired 3D growth. 19 To increase cell occupancy in the microwells, the bottom surface can be modified with cell adhesion molecules (e.g. fibronectin),20 but the utilization of cell adhesion molecules may lead to excessive interaction between the cells and the bottom surface, increasing the risk in cell growth in 2D. To establish a simple yet reliable approach to prepare cell spheroids, physically crosslinked hydrogels of poly(vinyl alcohol) (PVA) were microstructured here to generate MCSs (Scheme 1). PVA is a biocompatible materials approved by Food and Drug Administration (FDA)21 and can be micro-molded into an inverted pyramid-shaped microwell array. We investigated on the one hand, how the funnel-like shape of the well may be beneficial towards cell accumulation to maximize cell-cell interactions compared to the flat or round bottom of conventional microwells.


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On the other hand, we hypothesized that cells settling down at the tip of the well have no space to spread, which forces the entrapped cells to contact each other, and is therefore more beneficial the generation of cell spheroids. In addition, the weak interactions between the cells and PVA hydrogel substrates likely further prevent cells from attaching and spreading on the side walls, facilitating the formation of multicellular aggregates. More interestingly, PVA can be physically cross-linked to form hydrogels through repeated freezing-thawing without introducing extra cross-linking agent. The mechanical properties of PVA hydrogels can be easily tuned by changing the number of freezing-thawing cycles, which makes it possible to adjust the mechanical properties of the microwell according to the cultured cell type. 22 Due to the optimized design of shape and material of microwell array, no extra technology, e.g. microfluidics, is needed for the formation of cell spheroids, which is normally required in the recent of study of 3D cell culture.23

Scheme 1. Scheme of 2D cell settling on a flat hydrogel and the formation of 3D cell spheroids in the microstructured hydrogel. The size of cell spheroids can be varied by tuning cell culture


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time and initial seeding cell number. Spheroids generated after 5 days culture exhibited good cell viability and were used as tumor model to perform the anti-cancer drug testing. Using this platform the strategy was tested, the viability of cells and cell clusters was interrogated and the dependence of the MCS size on cell culture time and initially seeded cell number was determined. Finally, the applicability in drug testing was exemplarily investigated to highlight the potential of this platform approach for various applications in the biomedical field.

EXPERIMENTAL SECTION Materials. PVA powder (MW = 89,000 – 98,000 g/mol, hydrolysis > 99%) was purchased from Aldrich. Fluorescein diacetate (FDA) and Hoechst 33258 were purchased from Sigma-Aldrich, and propidium iodide (PI) was purchased from Carl Roth. Paraformaldehyde and Triton X-100 were purchased from VWR. Phalloidin-rhodamine was purchased from Invitrongen, Life Technologies. The cell counting assay kit (CCK8) was purchased from Dojindo, Japan. 6-Well cell culture plates were purchased from Sarstedt AG & Co. (Nürnbrecht, Germany). Throughout the whole study, water was purified using a Millipore Direct Q8 system (Millipore Advantage A10 system, Schwalbach, with Millimark Express 40 Filter, Merck, Germany) affording water with a resistivity of 18.2 MΩ cm. Preparation of PVA Hydrogel Microwell Arrays. A commercial AggreWell400Ex plate (STEMCELL Technologies, Canada), which has square pyramidal microwells with 400 µm side length, was employed as a template to first fabricate polydimethylsiloxane (PDMS) moulds. The degassed PDMS precursor mixed with the curing agent (10:1) was poured on the


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AggreWell400Ex chip and cured at 70°C for 2 h. The PDMS was peeled off to obtain PDMS moulds. Prior to the fabrication of microstructured PVA hydrogel, the PDMS mould was treated with O2 plasma (Plasma Prep2, Gala, Gabler Labor Instrumente GmbH, Germany) for 1 min to render the surface hydrophilic. The aqueous PVA solution (10 wt%) was then poured on the PDMS moulds. A PVA hydrogel was obtained by subjecting the aqueous polymer solutions to 4 times repeated freeze-thaw cycles, consisting of a 17 h freeze step at -20°C followed by a 7 h thawing step at 20°C. Finally, the microstructured PVA hydrogel was separated from PDMS template to obtain the microstructured hydrogel. Herein, PVA powers with three different molar masses of 13,000 - 23,000 g/mol, 31,000 - 50,000 g/mol, and 89,000 - 98,000 g/mol were tested for the fabrication of microwells. It was found that PVA hydrogel with 89,000 - 98,000 g/mol can preserve the pyramidal shape well, while the other two PVAs with 13,000 - 23,000 g/mol and 31,000 - 50,000 g/mol could hardly keep microwell structures. Therefore, PVA with 89,000 - 98,000 g/mol was chosen for preparing the microstructured hydrogel arrays. Measurement of Thermal Properties. Both the PVA hydrogel and solution with the same polymer concentration (10 wt%) were dried in an oven at 35°C at ambient pressure overnight prior to the DSC and ATR-FTIR measurements. The thermal properties (in terms of the heat of melting (∆Hm) and degree of crystallinity) of the PVA xerogel, obtained by drying the PVA hydrogel and PVA film formed by directly drying polymer solutions were characterized using Differential Scanning Calorimetry (DSC) analysis. The measurement was carried out using a TA instrument DSC Q1000. Both samples were heated from 10°C to 250°C with a heating rate of 10°C/min under a nitrogen flow. The ∆Hm values were determined from the melting peak of the DSC curves and the crystallinity of the samples was calculated by dividing the ∆Hm of the sample by the value of ∆Hm of 100% crystalline PVA (138.6 J/g).24


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Attenuated Total Internal Reflection Fourier Transform Infrared (ATR-FTIR) Spectroscopy. The samples for ATR-FTIR measurement were all dried in an oven at 35°C at ambient pressure overnight. ATR-FTIR spectra were recorded on a Bruker TENSOR27 using the diamond lens ATR module. The samples were scanned with a resolution of 0.5 cm-1 between 600 and 4000 cm-1. Optical Microscopy. To observe the microstructured PVA hydrogel in situ, a hydrogel sample placed upside down on a Petri dish was directly imaged by optical microscopy (Primovert, Carl Zeiss, Oberkochen, Germany). Scanning Electron Microscopy (SEM). To further observe details of the micro-wells as well as the hydrogel’s inner structure, SEM was used. The microstructured PVA hydrogel was dried in an oven at 35°C at ambient pressure overnight. For the observation of the interior structures of the PVA hydrogel, the sample was freeze dried. To observe cells cultured on the PVA hydrogels, the cells were fixed and dehydrated according to published protocols. 25 Before imaging, all samples were mounted on an aluminium stub and sputter coated with gold (8 – 10 nm). SEM images were taken using a CamScan microscope (CS24, USA). Cell Preparation. The PaTu 8988t cell line, a human pancreas adenocarcinoma cell line, was obtained from Dr. Jürgen Schnekenburger (Biomedical Technology Center of Medical Faculty Münster, Germany). The cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 5% fetal bovine serum (FBS), 5% hourse serum, 100 U mL-1 penicillin, 100 µg mL-1 streptomycin, and 2 × 10-3 M L-glutamine (all from ThermoFisher). The cells were cultured in a humidified incubator at 37°C and 5% CO2. In Vitro Cell Culture. The PVA hydrogels were placed into a 6-well cell culture plate and then cells suspended in 4 mL medium was pipetted into each well. Subsequently the cells were


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incubated at 37°C and 5% CO2 in a cell incubator. The cell culture medium was refreshed every two days. Live Dead Staining and Imaging. A fluorescent live-dead staining assay was used to visualize the viable cells and dead cells. 4 µL of FDA (10 mg mL-1) and 50 µL of PI (2 mg mL-1) were added in 5 mL of phosphate buffered saline (PBS) solution to prepare the assay solution. For staining cells, the cell culture medium was first removed and the hydrogel surface was washed with PBS solution. The assay solution (2 mL) was pipetted onto each hydrogel surface cultured with cells. After 5 min incubation in the dark, the staining solution was removed and then the hydrogel surface was washed with PBS solution. For fluorescence imaging, all the PVA hydrogel samples were placed upside down on a new Petri dish. The fluorescence images were obtained in a fluorescence microscope (Axiovert 135, Carl Zeiss, Oberkochen, Germany). The diameter of the cell aggregates was determined by using ZEN 2 (blue edition) software. Immunofluorescent Staining for Actin Cytoskeleton and Cell Nuclei. Immunofluorescent staining was carried out according to published protocols, using phalloidin-rhodamine and Hoechst 33238 as stains.26 The fluorescence images were obtained by a fluorescence microscope (Axiovert 135, Carl Zeiss, Oberkochen, Germany). For fluorescence imaging, all the PVA hydrogels were placed upside down on the petri dish. Anti-cancer Drug Testing. In the anti-cancer drug sensitivity test, spheroids were cultured with 5-fluorouracil (5-FU) for 3 days, which could irreversibly inhibit thymidylate synthase.27 5FU powder was first dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions with concentrations at 0, 0.1, 1, 10, and 100 mM, respectively. Then the stock solutions of 5-FU were diluted in cell culture medium at a volume ratio of 1 : 1000. The final volume fractions of DMSO were all 0.1v/v%, and concentrations of 5-FU were 0, 0.1, 1, 10, and 100 µM,


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respectively. On day 5 of cell spheroid culture, 4 mL of each concentration of 5-FU solution was added to each well. For the control group, cell culture medium without DMSO and 5-FU was used. The cell survival rate was measured by using a Cell Counting Kit-8 (CCK-8) assay and calculated according to: Survival rate (%) =

╳ 100 (%)

(equation 1)

where Asample is the measured absorbance at 450 nm for test sample, Ac is the absorbance at 450 nm for the negative control, and Ab is the absorbance at 450 nm for the blank (Scheme 2).

Scheme 2. Detailed explanation of Asample, Ac, and Ab in the CCK-8 assay. For the CCK-8 assay, the PVA hydrogel was put in a new 6-well plate and incubated in 2 mL of cell culture medium containing 100 µL CCK-8 solution for 5 h. 100 µL incubated medium was transferred into a well of a 96-well plate. The absorbance of the sample was measured at 450 nm using a microplate reader (Tecan SAFIRE, Tecan, Switzerland).

RESULTS AND DISCUSSION Preparation and Characterization of PVA Hydrogels. In this study, PVA hydrogels were prepared through four freeze-thaw cycles without any chemical crosslinking reaction (Figure S1). It is well known that aqueous PVA solutions can be transformed into a hydrogel via crystallite formation in repeated freeze-thaw cycles.28 During the freezing of the PVA solution, ice created


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in the amorphous region induces the growth of polymer crystallites, acting as junction knots of the network and thus facilitates the formation of hydrogel.29 ATR-FTIR measurements were firstly undertaken to qualitatively investigate the crystallization of PVA in the hydrogel (Figure 1a). The spectrum of the PVA xerogel shows not only the characteristic peaks of PVA (the corresponding peak assignments are shown in Figure 1b ), but also an important and characteristic band at 1142 cm-1 related to the C-O stretching, which verifies the crystalline structure of the sample.30 This band at 1142 cm-1 was also detected for PVA films directly dried from PVA aqueous solution, suggesting that crystallization took place in the films. DSC measurements were further used to analyse the crystallinity of the PVA films and xerogels (Figure 1c). The sharp peaks around 228°C in the DSC curves correspond to the melting temperature of the PVA crystallites. The crystallinity of PVA films and xerogels calculated from the acquired ∆Hm values are shown in Figure 1d. The results confirm that repeated freeze-thaw treatment significantly increases the crystallinity of PVA xerogels compared to that of PVA films.


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Figure 1. (a) ATR-FTIR spectra of PVA film and xerogel. (b) Characteristic FTIR peaks of the samples and their assignments. (c) DSC curves of PVA film and xerogel. (d) Crystallinities of PVA film and xerogel calculated from ∆Hm values. Characterization of Morphology of Microstructured PVA Hydrogel. A commercial AggreWell400Ex plate containing pyramid-shaped microwells with 400 µm side length was used as template for patterning PVA hydrogel. As shown in Figure 2a-c, PDMS moulds fabricated from AggreWell400Ex plate consist of a high-density array of square-pyramids with about 400 µm width and 250 µm height. The morphology of the microstructured PVA hydrogel was analyzed using optical microscopy performed on hydrated hydrogel samples. The corresponding microstructured PVA hydrogel molded from PDMS mould was composed of square-pyramidal wells with 400 µm width and 250 µm depth (Figure 2d-e). After drying the hydrogel, the typical shape of the microwell could be observed under SEM as well as the concomitant deformation of wells due to the concomitant hydrogel shrinkage (Figure 2f). The inner structure of the PVA


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hydrogel exhibits an irregular porous network structure along with many fibers (inset in Figure 2f). These pores are mainly generated by the ice crystals formed during the freeze-thaw process, which result in the space between crosslinking points.31

Figure 2. Optical microscopy images of (a, b) PDMS mould and (d, e) microstructured PVA hydrogel. The samples of Figure 2a and 2d were observed upside down. Figure 2b and 2e are cross-sections of the PMDS mould and the microstructured PVA hydrogel, respectively. SEM images of (c) PDMS mould and (f) microstructured PVA hydrogel after drying. Spheroid Formation on Microstructured Hydrogel Surface. To investigate the cell behavior on the microstructured and non-patterned PVA hydrogels, a pancreatic cancer cell line (PaTu 8988t) was chosen as the test cell line.32 Microstructured and flat PVA hydrogels (surface areas of 9.6 cm2) were placed in 6-well plates to support cell growth. After the addition of cell suspension (2.0 × 105 cells in 4 mL cell culture medium) to the PVA hydrogels, the cells settled toward the hydrogel surface under the influence of gravity. The antifouling property of PVA resulted in only a small number of cells that adhered and proliferated on the non-patterned flat


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hydrogel (Figure S2). The cells on the flat hydrogel surface grew in two dimensions, and no cell spheroid was observed. By contrast, cells on the microstructured hydrogel were rapidly entrapped in the pyramid-shaped microwell and then accumulated at the central tip (Figure 3a), which allowed cells to grow in all directions (Figure 3b-e). Typical cell spheroids formed after 3 days culture with diameters of 82 ± 15 µm. The cell spheroids gradually grew with increasing culture time. The diameter of the cell spheroids reached 157 ± 25 µm after 5 days of culture, which may be expected to further increase if the incubation is continued (Figure 3f). As can be seen, the size of cell spheroid was controlled by tuning the cell culture time. However, the distribution of cell spheroid size gradually became broader with increasing culture time (Figure S3). The entirely different cell behavior on the microstructured and non-patterned PVA hydrogels suggests that the pyramid-shaped microwells play a crucial role in the formation of 3D MCS. From the results of live-dead staining (Figure 3e and Figure S2f), the live cells on the microstructured hydrogel were much higher than those on the flat PVA hydrogel. Despite the hydrophilic nature of PVA, which renders its surface unsuitable for cell attachment, the pyramidshaped microwell promotes the initial accommodation of cells and final number of proliferated cells. Due to the low cell adhesion on the PVA surface, the entrapped cells in the microwells do not attach and spread on the wall surface of wells, but prefer to attach to each other and form subsequently the desired cell spheroids.


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Figure 3. Fluorescence microscopy images of PaTu 8988t cells cultured in PVA microwells at an initially seeded cell number of 2.0 × 105 following a live-dead stain after different culture times. Live cells were stained with green and dead cells were stained with red. Culture times: (a) 1 day, (b) 2 days, (c) 3 days, (d) 4 days, (e) 5 days. (f) The diameter of the cell spheroids increased with increasing culture time. The cell spheroid shape was approximated as round shape to determine the diameter, which was measured using ZEN 2 (blue edition) software. Error bar: standard error (n = 40). The cell morphology was investigated by SEM in detail. Most microwells were found to be occupied by cell spheroids (Figure 4a). The ratio of microwells occupied by cells reached 72 ± 6% at an initial cell number of 2.0 × 105. For the PVA hydrogel prepared from the 6-well plates


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in this work, the number of microwells is ~4700. Therefore, one microstructured hydrogel produced up to about 3400 cell spheroids, which allows for massive production of MCSs. Cell-cell contacts on the spheroids were clearly observed in the magnified SEM images (Figure 4b). The cells remained to be round in shape during the aggregation process, suggesting they did not prefer to attach to the hydrogel surface because of the weak interaction between PVA hydrogel and the cells. The weak cell-substrate interactions hence facilitate the formation of multicellular aggregates. 33 The ratio of microwells occupied by PaTu 8988t cells was influenced by initial cell number (Figure 3c). Microwell occupancy by cells was only 9 ± 5% at an initial cell number of 1 × 104 which can be gradually promoted to 72 ± 6% with increasing seeded cell number of 2.0 × 105. Compared to microstructured hydrogels, the non-patterned hydrogels can only support 2D cell growth (Figure 4d and e), allowing cells to attach and proliferate on the gel surface. The result of the CCK-8 assay (Figure 4f) displayed that the absorbance at 450 nm for 3D MCSs is 9 times higher than that for 2D cell culture, which confirmed that the number of live cells on the microstructured hydrogel was much higher than that on the flat hydrogel due to the presence of pyramid-shaped microwells.


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Figure 4. (a and b) SEM image of 3D MCSs generated on the microstructured PVA hydrogels. (c) The ratio of microwells occupied by PaTu 8988t cells increased with increasing initial cell number. (d and e) SEM images of cells cultured on the non-patterned PVA hydrogels. (f) CCK-8 assay results of 3D MCSs (absorbance ≈ 1.10 ± 0.09) and cells cultured in 2D (absorbance ≈ 0.12 ± 0.01). The higher absorbance at 450 nm confirms a higher live cell number on the 3D samples. Not only the microwell occupancy, but also the cell aggregate diameter was found to highly depend on the initially seeded cell number. The average size of the cell spheroids was determined after 5 days culture. As shown in Figure 5, there is no cell spheroid found in the microwells at a seeded cell number of 1 × 104. With an increase in number of seeded cell, cell spheroids formed in the microwells, meanwhile, the corresponding diameter of cell spheroid increased. Typically, cell spheroids with a mean diameter of 157 ± 25 µm were observed in the microwells. The micro-patterned PVA hydrogel was still stable after immersion for 10 days in


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cell medium (Figure S4) or even longer time, which indicates the possibility to do long-term cell culture.

Figure 5. Fluorescence microscopy images of PaTu 8988t cells cultured in PVA microwells for 5 days for different initially seeded cell numbers following a live-dead stain. Live cells were stained with green and dead cells were stained with red. Initial seeded cell numbers: (a) 1 × 104 cells, (b) 2.5 × 104 cells, (c) 5.0 × 104 cells, (d) 1.0 × 105 cells, (e) 2.0 × 105 cells. (f) The diameter of the cell spheroid increased with increasing initial cell number. The cell spheroid shape was approximated as round shape to determine the diameter, which was measured using ZEN 2 (blue edition) software. Error bar: standard error (n = 40). Immunofluorescence staining showed that F-actin was expressed in the cells in the both 2D cell layers and 3D spheroids. For 2D cell culture, only a small part of the cells showed a typical 2D spread-out morphology (as pointed by arrows in Figure 6). Most cells on the flat hydrogel


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displayed a rounded morphology. The actin filaments of the cells were mainly located around the edges of the cells with a cortical distribution. This is attributed to the mentioned low interaction between the cells and the PVA hydrogel surface. By contrast, the cells on the microstructured hydrogels presented a clustered morphology, suggesting the formation of spheroids.

Figure 6. Immunofluorescent images of PaTu 8988t cultured in 2D and grown into 3D spheroids. The cell nuclei and the actin cytoskeleton were stained in blue and red, respectively. Anticancer Drug Sensitivity Tests. To unravel the impact of cell-cell contacts on the sensitivity of the cells vs. drugs, PaTu 8988t cells were treated after 5 day cell culture with the classic anticancer drug 5-fluorouracil (5-FU). The drug was administered in different concentrations to both spheroid and 2D culture groups. Figure 7 shows the results of the livedead staining and the corresponding survival rates of PaTu 8988t cells after exposure to 5-FU for 72 h. Firstly, 0.1 v/v% DMSO in culture medium did not influence the cell viability and the


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survival rates for both 2D cell culture and 3D MCSs were higher than 97%. As the concentration of 5-FU increased, the percentage of live cell decreased rapidly for both 3D and 2D cell culture conditions. At the same drug concentration, the cell viability in spheroids was much higher than that for cells cultured in 2D (Figure 7b). This effect can be attributed to the longer time needed for the drug to diffuse and penetrate into the spheroids.34 Furthermore, 5-FU specifically targets proliferating cells.35 3D multicellular spheroids have an external proliferating zone and internal quiescent zone caused by the lack of nutrients and gas (e.g. O2, CO2) exchange.36 Thus, 5-FU will not kill the quiescent cells in the spheroids. By contrast, cells in 2D culture are mostly proliferating cells, which are inhibited by 5-FU more effectively. Particularly, at a concentration of 100 µM, the survival rate for 2D cells was only 10%, while it achieved 61% for 3D cell spheroids. Except for the two points mentioned above, the differences in cellular response between 2D cultures and 3D spheroids may be also associated with other aspects, such as different gene and surface receptor expressions, different local pH values, etc.37 In general, 3D spheroids provide a more physiologically relevant model for better prediction of drug effects and the data confirm that cell aggregates are typically more resistant to chemotherapies compare to cells cultured in 2D.38


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Figure 7. Anticancer drug testing of microstructured PVA hydrogel generated PaTu 8988t spheroids. (a) Fluorescence microscopy images of live-dead staining experiments of PaTu 8988t spheroids and 2D cell layer after 72 h of anticancer drug 5-FU treatment. Live and dead cells were stained with green and red, respectively. (b) Bar graph of the survival rate of cells after 72 h of drug treatment. The results were calculated using the CCK-8 assay. The statistical analysis shows a significant difference between the 3D spheroids and 2D culture for different drug concentrations (***p < 0.0001, **p < 0.001, and *p 0.05) different. Error bars: standard error (n = 3). (c) Schematic diagram of proliferating zone,


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quiescent zone, and necrotic zone in a 3D spheroid, with schematic representation of nutrition, O2, CO2, and wastes exchange, and limited diffusion and penetration of 5-FU drug molecules. Release of Cell Spheroids. Once the spheroids have been formed in the microstructured hydrogel microwells, they can be released from PVA hydrogel without any enzyme treatment simply by rinsing the hydrogel surface (Figure 8). The low adhesion of the cells to the PVA surface facilitates this simple cell release. The released cell aggregates in the cell medium also remained intact after repeated rinsing. This demonstrates that the cells in the aggregates are indeed attached to each other by cell-cell adhesions, which facilitates cellular communication, and are not simply accumulated in the microwells. After release, the cell spheroids were transferred to a new 2D cell culture plate. After being cultured for 24 h, the cell spheroids were partially disassembled (Figure S5). Especially, the outside cells attached and spread on the substrate, presenting an irregular shape, which suggests these cells retained the capability to migrate.39


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Figure 8. (a) Schematic illustration of cell spheroids release by rinsing the microwell surface. (b) Fluorescence microscopy images of live-dead stained cells in the microwells before rinsing. (c) Few cells were found to remain in the microwells after flushing. (d) Intact cell spheroids reelased into the medium. CONCLUSION In conclusion, a novel microstructured PVA hydrogel with inverted pyramid-shaped microwells can be readily used for massive MCSs generation. The size of cell spheroids can be controlled by the number of cells seeded in the well or by the culture time. The cells in the formed MCSs kept a high viability for at least 8 days of cell culture. The cell spheroids can be harvested by rinsing with cell medium, faciliated by the low adhesion of the cells to the PVA surface, which avoids the complications of enzyme-based release treatments. The generated MCSs showed higher resistance to the proliferation-inhibiting drug 5-FU compared to cells cultured in 2D, which is


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very useful for drug testing. Moreover, this PVA hydrogel microwell system makes it possible to pre-embed specific biomolecules (e.g. growth factors, inducing factors, and proteins) to support 3D cell growth, not only tumor cells, but even stem cells. In the future, test of different drugs on the PVA hydrogel platform can be done with large number of arrays by individually separating cell spheroids in different microwells, as described by reported high throughput screening.40 It is expected that this microstructured PVA hydrogel-based spheroid production provides a straightforward approach to obtain MCSs, which affords viable spheroids for potential application in drug screening, tissue engineering, pharmacology, and relative biological research.

ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Fabrication method to obtain the microstructured PVA hydrogel; Fluorescence microscopy data of live-dead staining of 2D cell culture on flat PVA hydrogel; size distribution of cell spheroids and optical microscopy images of released cell spheroids re-seeded on a new 2D cell culture plate. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (X. Q. Dou)


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*E-mail: [email protected] (H. Schönherr) Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Funding Sources This work was supported by the Alexander von Humboldt Foundation (postdoc stipend to X. Q. Dou), the European Research Council (ERC grant no. 279202), and the University of Siegen. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We thank Dipl.-Biol. Sabine Wenderhold-Reeb and Dipl.-Ing. Gregor Schulte for their support, as well as Dr. Jürgen Schnekenburger (Biomedical Technology Center of Medical Faculty Münster, Germany), who kindly provided the PaTu 8988t cell line, and Prof. Dr. Ulrich Jonas, Frau Frank (Macromolecular Chemistry Department Chemistry – Biology, University of Siegen), who kindly granted access to DSC and ATR-FTIR for PVA characterization. REFERENCES

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Table of Contents Graphic and Synopsis

Physically crosslinked poly(vinyl alcohol) (PVA) hydrogels structured with inverted pyramidshaped microwells were fabricated to generate three-dimensional (3D) multicellular cell spheroids (MCSs), which provide a better mimick of in vitro cell models for various biomedical studies, including drug testing, pathological study, and tissue engineering.


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Three-Dimensional Microstructured Poly(vinyl alcohol) Hydrogel

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