Three-Dimensional Microstructured Poly (vinyl alcohol) Hydrogel

Nov 21, 2017 - A PVA hydrogel was obtained by subjecting the aqueous polymer solutions to 4 times repeated freeze–thaw cycles, consisting of a 17 h ...
0 downloads 0 Views 2MB Size
Download PDF
Article Cite This: Biomacromolecules 2018, 19, 158−166

pubs.acs.org/Biomac

Three-Dimensional Microstructured Poly(vinyl alcohol) Hydrogel Platform for the Controlled Formation of Multicellular Cell Spheroids Xiaoqiu Dou,* Ping Li, and Holger Schönherr* Physical Chemistry I and Research Center of Micro and Nanochemistry and Engineering (Cμ), Department of Chemistry and Biology, University of Siegen, Adolf-Reichwein-Str. 2, 57076, Siegen, Germany S Supporting Information *

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 welldefined 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 accumulated at the central tip of the wells and then gradually grew 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 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

On the basis of the merits of MCSs, a variety of techniques have been explored to promote the formation of uniform-sized cell spheroids, including the use of microtextured surfaces,8 hanging drop technique,9 chemical micropatterns,10 thermoresponsive polymers,11 cell encapsulation in hydrogels12 or rotary bioreactors,13 and so on.14,15 Recently, taking advantage 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

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 submonolayers 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 and 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 © 2017 American Chemical Society

Received: September 17, 2017 Revised: November 19, 2017 Published: November 21, 2017 158

DOI: 10.1021/acs.biomac.7b01345 Biomacromolecules 2018, 19, 158−166

Article

Biomacromolecules

this platform approach for various applications in the biomedical field.

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 cross-linked hydrogels of poly(vinyl alcohol) (PVA) were microstructured here to generate MCSs (Scheme 1). PVA is a biocompatible material approved by

■

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. Six-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) molds. The degassed PDMS precursor mixed with the curing agent (10:1) was poured on the AggreWell400Ex chip and cured at 70 °C for 2 h. The PDMS was peeled off to obtain PDMS molds. Prior to the fabrication of microstructured PVA hydrogel, the PDMS mold 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 molds. 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 the PDMS template to obtain the microstructured hydrogel. Herein, PVA powders with three different molar masses of 13,000−23,000, 31,000−50,000, 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 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 (ΔHm) 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 Instruments DSC Q1000. Both samples were heated from 10 to 250 °C with a heating rate of 10 °C/min under a nitrogen flow. The ΔHm values were determined from the melting peak of the DSC curves, and the crystallinity of the samples was calculated by dividing the ΔHm of the sample by the value of ΔHm of 100% crystalline PVA (138.6 J/g).24 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 microwells 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.

Scheme 1. Scheme of a 2D Cell Settling on a Flat Hydrogel and the Formation of 3D Cell Spheroids in the Microstructured Hydrogela

a

The size of cell spheroids can be varied by tuning cell culture time and initial seeding cell number. Spheroids generated after 5 days of culture exhibited good cell viability and were used as a tumor model to perform the anti-cancer drug testing.

Food and Drug Administration (FDA)21 and can be micromolded into an inverted pyramid-shaped microwell array. We investigated on the one hand how the funnel-like shape of the well may be beneficial toward cell accumulation to maximize cell−cell interactions compared to the flat or round-bottom of conventional microwells. 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 for 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 crosslinking 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 the shape and material of the microwell array, no extra technology, e.g., microfluidics, is needed for the formation of cell spheroids, which is normally required in the recent study of 3D cell culture.23 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 159

DOI: 10.1021/acs.biomac.7b01345 Biomacromolecules 2018, 19, 158−166

Article

Biomacromolecules 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 aluminum 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 sixwell cell culture plate, and then, cells suspended in 4 mL of medium were pipetted into each well. Subsequently, the cells were incubated at 37 °C and 5% CO2 in a cell incubator. The cell culture medium was refreshed every 2 days. Live Dead Staining and Imaging. A fluorescent live−dead staining assay was used to visualize the viable cells and dead cells. Four 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 of incubation in the dark, the staining solution was removed and then the hydrogel surface was washed with PBS solution. For fluorescence imaging, all of the PVA hydrogel samples were placed upside down on a new Petri dish. The fluorescence images were obtained by 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 of the PVA hydrogels were placed upside down on the Petri dish. Anticancer Drug Testing. In the anticancer drug sensitivity test, spheroids were cultured with 5-fluorouracil (5-FU) for 3 days, which could irreversibly inhibit thymidylate synthase.27 5-FU 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 0.1 v/v%, and concentrations of 5-FU were 0, 0.1, 1, 10, and 100 μM, 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 Kit8 (CCK-8) assay and calculated according to

Survival rate (%) =

A sample − A b Ac − A b

× 100 (%)

For the CCK-8 assay, the PVA hydrogel was put in a new six-well plate and incubated in 2 mL of cell culture medium containing 100 μL of 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 cross-linking 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 in the amorphous region induces the growth of polymer crystallites, acting as junction knots of the network and thus facilitating the formation of hydrogel.29 ATR-FTIR measurements were first 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 analyze 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 crystallinities of PVA films and xerogels calculated from the acquired ΔHm 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. Characterization of Morphology of Microstructured PVA Hydrogel. A commercial AggreWell400Ex plate containing pyramid-shaped microwells with 400 μm side length was used as a template for patterning PVA hydrogel. As shown in Figure 2a−c, PDMS molds fabricated from the 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 mold 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 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 cross-linking points.31 Spheroid Formation on Microstructured Hydrogel Surface. To investigate the cell behavior on the microstructured and nonpatterned 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 six-well plates to support cell growth. After the addition of cell suspension (2.0 × 105 cells in 4 mL of cell

(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

160

DOI: 10.1021/acs.biomac.7b01345 Biomacromolecules 2018, 19, 158−166

Article

Biomacromolecules

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 ΔHm values.

Figure 2. Optical microscopy images of (a, b) PDMS mold and (d, e) microstructured PVA hydrogel. The samples of parts a and d were observed upside down. Parts b and e are cross sections of the PMDS mold and the microstructured PVA hydrogel, respectively. SEM images of (c) PDMS mold and (f) microstructured PVA hydrogel after drying.

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 nonpatterned flat 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 of 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 the 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 nonpatterned 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 pyramid-shaped 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. 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 six-well plates 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. 161

DOI: 10.1021/acs.biomac.7b01345 Biomacromolecules 2018, 19, 158−166

Article

Biomacromolecules

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 a round shape to determine the diameter, which was measured using ZEN 2 (blue edition) software. Error bar: standard error (n = 40).

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 nonpatterned 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.

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 nonpatterned hydrogels can only support 2D cell growth (Figure 4d and e), allowing cells to attach and proliferate on the gel

Cell−cell contacts on the spheroids were clearly observed in the magnified SEM images (Figure 4b). The cells remained 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 162

DOI: 10.1021/acs.biomac.7b01345 Biomacromolecules 2018, 19, 158−166

Article

Biomacromolecules

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 a round shape to determine the diameter, which was measured using ZEN 2 (blue edition) software. Error bar: standard error (n = 40).

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.

after 5 days of 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 the number of seeded cells, 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 micropatterned PVA hydrogel was still stable after immersion for 10 days in cell medium (Figure S4)

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 pyramidshaped microwells. 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 163

DOI: 10.1021/acs.biomac.7b01345 Biomacromolecules 2018, 19, 158−166

Article

Biomacromolecules

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 < 0.01), whereas the fractions of live cells treated by only 0.1 v/v% DMSO on 3D cell spheroids and 2D cell culture are not significantly (ns: p > 0.05) different. Error bars: standard error (n = 3). (c) Schematic diagram of the proliferating zone, quiescent zone, and necrotic zone in a 3D spheroid, with a schematic representation of nutrition, O2, CO2, and waste exchange, and limited diffusion and penetration of 5-FU drug molecules.

Figure 8. (a) Schematic illustration of cell spheroid 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 released into the medium.

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.

or an even longer time, which indicates the possibility to do long-term cell culture. Immunofluorescence staining showed that F-actin was expressed in the cells in 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 out by arrows in Figure 6). Most cells on the flat hydrogel displayed a rounded 164

DOI: 10.1021/acs.biomac.7b01345 Biomacromolecules 2018, 19, 158−166

Article

Biomacromolecules 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 live−dead staining and the corresponding survival rates of PaTu 8988t cells after exposure to 5-FU for 72 h. First, 0.1 v/v% DMSO in culture medium did not influence the cell viability and the 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, 5FU 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 also be 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 compared to cells cultured in 2D.38 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

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, testing of different drugs on the PVA hydrogel platform can be done with a 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 hydrogelbased 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

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.7b01345. 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 reseeded on a new 2D cell culture plate (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Holger Schönherr: 0000-0002-5836-5569 Author Contributions

X.D. and P.L. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS 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. This work was supported by the Alexander von Humboldt Foundation (postdoc stipend to X.D.), the European Research Council (ERC grant no. 279202), and the University of Siegen.

■

CONCLUSION In conclusion, a novel microstructured PVA hydrogel with inverted pyramid-shaped microwells can be readily used for massive MCS 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 very useful for drug testing. Moreover, this PVA

■

REFERENCES

(1) Fukuda, J.; Khademhosseini, A.; Yeh, J.; Eng, G.; Cheng, J. J.; Farokhzad, O. C.; Langer, R. Microstructured Cell Co-Cultures Using Layer-by-Layer Deposition of Extracellular Matrix Components. Biomaterials 2006, 27, 1479−1486. (2) Fukuda, J.; Khademhosseini, A.; Yeo, Y.; Yang, X. Y.; Yeh, J.; Eng, G.; Blumling, J.; Wang, C. F.; Kohane, D. S.; Langer, R. Micromolding of Photocrosslinkable Chitosan Hydrogel for Spheroid Microarray and Co-cultures. Biomaterials 2006, 27, 5259−5267.

165

DOI: 10.1021/acs.biomac.7b01345 Biomacromolecules 2018, 19, 158−166

Article

Biomacromolecules (3) Bernard, A. B.; Lin, C. C.; Anseth, K. S. A Microwell Cell Culture Platform for the Aggregation of Pancreatic β-Cells. Tissue Eng. Tissue Eng., Part C 2012, 18, 583−592. (4) Weiswald, L. B.; Bellet, D.; Dangles-Marie, V. Spherical Cancer Models in Tumor Biology. Neoplasia 2015, 17, 1−15. (5) Mohr, J. C.; de Pablo, J. J.; Palecek, S. P. 3-D Microwell Culture of Human Embryonic Stem Cells. Biomaterials 2006, 27, 6032−6042. (6) Cordey, M.; Limacher, M.; Kobel, S.; Taylor, V.; Lutolf, M. P. Enhancing the Reliability and Throughput of Neurosphere Culture on Hydrogel Microwell Arrays. Stem Cells 2008, 26, 2586−2594. (7) Watanabe, K.; Kamiya, D.; Nishiyama, A.; Katayama, T.; Nozaki, S.; Kawasaki, H.; Watanabe, Y.; Mizuseki, K.; Sasai, Y. Directed Differentiation of Telencephalic Precursors from Embryonic Stem Cells. Nat. Neurosci. 2005, 8, 288−296. (8) Shin, H. S.; Kook, Y. M.; Hong, H. J.; Kim, Y. M.; Koh, W. G.; Lim, J. Y. Functional Spheroid Organization of Human Salivary Gland Cells Cultured on Hydrogel-Microstructured Nanofibrous Microwells. Acta Biomater. 2016, 45, 121−132. (9) Lin, B. J.; Miao, Y.; Wang, J.; Fan, Z. X.; Du, L. J.; Su, Y. S.; Liu, B. C.; Hu, Z. Q.; Xing, M. Surface Tension Guided Hanging-Drop: Producing Controllable 3D Spheroid of High-Passaged Human Dermal Papilla Cells and Forming Inductive Microtissues for HairFollicle Regeneration. ACS Appl. Mater. Interfaces 2016, 8, 5906−5919. (10) Liu, T. Q.; Yang, C. T.; Dieguez, L.; Denman, J. A.; Thierry, B. Robust and Flexible Fabrication for Tumor Spheroid Preparation. ACS Appl. Mater. Interfaces 2014, 6, 10162−10171. (11) Shimada, N.; Saito, M.; Shukuri, S.; Kuroyanagi, S.; Kuboki, T.; Kidoaki, S.; Nagai, T.; Maruyama, A. Reversible Monolayer/Spheroid Cell Culture Switching by UCST-Type Thermoresponsive Ureido Polymers. ACS Appl. Mater. Interfaces 2016, 8, 31524−31529. (12) Wang, D. D.; Cheng, D.; Guan, Y.; Zhang, Y. J. Thermoreversible Hydrogel for In Situ Generation and Release of HepG2 Spheroids. Biomacromolecules 2011, 12, 578−584. (13) Nyberg, S. L.; Hardin, J.; Amiot, B.; Argikar, U. A.; Remmel, R. P.; Rinaldo, P. Rapid Large-Scale Formation of Porcine Hepatocyte Spheroids in a Novel Spheroid Reservoir Bioartificial Liver. Liver Transplant. 2005, 11, 901−910. (14) Chen, K. J.; Wu, M. X.; Guo, F.; Li, P.; Chan, C. Y.; Mao, Z. M.; Li, S. X.; Ren, L. Q.; Zhang, R.; Huang, T. J. Rapid Formation of SizeControllable Multicellular Spheroids via 3D Acoustic Tweezers. Lab Chip 2016, 16, 2636−2643. (15) Estrada, M. F.; Rebelo, S. P.; Davies, E. J.; Pinto, M. T.; Pereia, H.; Santo, V. E.; Smalley, M. J.; Barry, S. T.; Gualda, E. J.; Alves, P. M.; Anderson, E.; Brito, C. Modelling the Tumour Microenvironment in Long-Term Microencapsulated 3D Co-Cultures Recapitulates Phenotypic Features of Disease Progression. Biomaterials 2016, 78, 50−61. (16) Zorlutuna, P.; Annabi, N.; Camci-Unal, G.; Nikkhah, M.; Cha, J. M.; Nichol, J. W.; Manbachi, A.; Bae, H.; Chen, S.; Khademhosseini, A. Microfabricated Biomaterials for Engineering 3D Tissues. Adv. Mater. 2012, 24, 1782−1804. (17) Charnley, M.; Textor, M.; Khademhosseini, A.; Lutolf, M. Integration Column: Microwell Arrays for Mammalian Cell Culture. Integr. Biol. 2009, 1, 625−634. (18) Liu, T. Q.; Winter, M.; Thierry, B. Quasi-spherical Microwells on Superhydrophobic Substrates for Long Term Culture of Multicellular Spheroids and High Throughput Assays. Biomaterials 2014, 35, 6060−6068. (19) Lilge, I.; Jiang, S. Y.; Schönherr, H. Long-Term Stable Poly(acrylamide) Brush Modified Transparent Microwells for Cell Attachment Studies in 3D. Macromol. Biosci. 2017, 17, 1600451. (20) Ochsner, M.; Dusseiller, M. R.; Grandin, H. M.; Luna-Morris, S.; Textor, M.; Vogel, V.; Smith, M. L. Micro-well Arrays for 3D Shape Control and High Resolution Analysis of Single Cells. Lab Chip 2007, 7, 1074−1077. (21) Chaouat, M.; Le Visage, C.; Baille, W. E.; Escoubet, B.; Chaobet, F.; Mateescu, M. A.; Letourneur, D. A Novel Cross-linked Poly(vinyl alcohol) (PVA) for Vascular Grafts. Adv. Adv. Funct. Mater. 2008, 18, 2855−2861.

(22) Kim, T. H.; An, D. B.; Oh, S. H.; Kang, M. K.; Song, H. H.; Lee, J. H. Creating Stiffness Gradient Polyvinyl Alcohol Hydrogel Using a Simple Gradual Freezing-Thawing Method to Investigate Stem Cell Differentiation Behaviors. Biomaterials 2015, 40, 51−60. (23) Chen, Y. C.; Lou, X.; Zhang, Z. X.; Ingram, P.; Yoon, E. HighThroughput Cancer Cell Sphere Formation for Characterizing the Efficacy of Photo Dynamic Therapy in 3D Cell Cultures. Sci. Rep. 2015, 5, 12175. (24) Peppas, N. A.; Hansen, P. J. Crystallization Kinetics of Poly(vinyl alcohol). J. Appl. Polym. Sci. 1982, 27, 4787−4794. (25) Lilge, I.; Jiang, S. Y.; Wesner, D.; Schönherr, H. The Effect of Size and Geometry of Poly(acrylamide) Brush-Based Micropatterns on the Behavior of Cells. ACS Appl. Mater. Interfaces 2016, 8, 23591− 23603. (26) Dou, X. Q.; Li, P.; Jiang, S. Y.; Bayat, H.; Schönherr, H. Bioinspired Hierarchically Structured Surfaces for Efficient Capture and Release of Circulating Tumor Cell. ACS Appl. Mater. Interfaces 2017, 9, 8508−8518. (27) Longley, D. B.; Harkin, D. P.; Johnston, P. G. 5-Fluorouracil: Mechanisms of Action and Clinical Strategies. Nat. Rev. Cancer 2003, 3, 330−338. (28) Stauffer, S. R.; Peppas, N. A. Poly(vinyl alcohol) Hydrogels Prepared by Freezing-Thawing Cyclic Processing. Polymer 1992, 33, 3932−3936. (29) Yokoyama, F.; Masada, I.; Shimamura, K.; Ikawa, T.; Monobe, K. Morphology and Structure of Highly Elastic Poly(vinyl alcohol) Hydrogel Prepared by Repeated Freezing-and-Melting. Colloid Polym. Sci. 1986, 264, 595−601. (30) Tretinnikov, O. N.; Zagorskaya, S. A. Determination of the Degree of Crystallinity of Poly(vinly alcohol) by FTIR Spectroscopy. J. Appl. Spectrosc. 2012, 79, 521−526. (31) Huang, M.; Cai, D. D.; Liu, Y. H.; Sun, J.; Wang, J. J.; Qin, C. X.; Dai, L. X.; Kazuo, Y. Investigation of a-PVA/s-PVA Hydrogels Prepared by Freezing-Thawing Method. Fibers Polym. 2012, 13, 955− 962. (32) Elsässer, H. P.; Lehr, U.; Agricola, B.; Kern, H. F. Establishment and Characterisation of Two Cell Lines with Different Grade of Differentiation Derived from One Primary Human Pancreatic Adenocarcinoma. Virchows Archiv B Cell Pathol. 1992, 61, 295−306. (33) Zhao, Z. Q.; Gu, J. J.; Zhao, Y. N.; Guan, Y.; Zhu, X. X.; Zhang, Y. J. Hydrogel Thin Film with Swelling-Induced Wrinkling Patterns for High-Throughput Generation of Multicellular Spheroids. Biomacromolecules 2014, 15, 3306−3312. (34) Wan, X.; Li, Z. H.; Ye, H.; Cui, Z. F. Three-dimensional Perfused Tumor Spheroid Model for Anti-cancer Drug Screening. Biotechnol. Lett. 2016, 38, 1389−1395. (35) Tung, Y. C.; Hsiao, A. Y.; Allen, S. G.; Torisawa, Y. S.; Ho, M.; Takayama, S. High-throughput 3D Spheroid Culture and Drug Testing Using a 384 Hanging Drop Array. Analyst 2011, 136, 473− 478. (36) Zanoni, M.; Piccinini, F.; Arienti, C.; Zamagni, A.; Santi, S.; Polico, R.; Bevilacqua, A.; Tesei, A. 3D Tumor Spheroid Models for in vitro Therapeutic Screening: a Systematic Approach to Enhance the Biological Relevance of Data Obtained. Sci. Rep. 2016, 6, 19103. (37) Edmondson, R.; Broglie, J. J.; Adcock, A. F.; Yang, L. J. ThreeDimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors. Assay Drug Dev. Technol. 2014, 12, 207−218. (38) Torisawa, Y. S.; Takagi, A.; Shiku, H.; Matsue, T. A Multicellular Spheroid-Based Drug Sensitivity Test by Scanning Electrochemical Microscopy. Oncol. Rep. 2005, 13, 1107−1112. (39) Lee, J.; Cuddihy, M. J.; Cater, G. M.; Kotov, N. A. Engineering Liver Tissue Spheroids with Inverted Colloidal Crystal Scaffolds. Biomaterials 2009, 30, 4687−4694. (40) Ankam, S.; Teo, B. K.; Kukumberg, M.; Yim, E. K. High throughput screening to investigate the interaction of stem cells with their extracellular microenvironment. Organogenesis 2013, 9, 128−142.

166

DOI: 10.1021/acs.biomac.7b01345 Biomacromolecules 2018, 19, 158−166