Dual-Phase, Surface Tension-Based Fabrication Method for

Mar 11, 2014 - Design of spherically structured 3D in vitro tumor models -Advances and prospects. L.P. Ferreira , V.M. Gaspar , J.F. Mano. Acta Biomat...
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Dual-Phase, Surface Tension-Based Fabrication Method for Generation of Tumor Millibeads Shantanu Pradhan,† Chloe S. Chaudhury,‡,§ and Elizabeth A. Lipke*,† †

Department of Chemical Engineering, Auburn University, Auburn, Alabama 36849, United States Department of Cellular and Molecular Biosciences, Auburn University, Auburn, Alabama 36849, United States

‡

S Supporting Information *

ABSTRACT: Numerous methods have been developed for the fabrication of poly(ethylene glycol)-based hydrogel microstructures for drug-delivery and tissue-engineering applications. However, present methods focus on the fabrication of submicrometer scale hydrogel structures which have limited applications in creating larger tissue constructs, especially in recreating cancer tissue microenvironments. We aimed to establish a platform where cancer cells can be cultured in a three-dimensional (3D) environment, which closely replicates the native cancer microenvironment and facilitates efficient testing of anticancer drugs. This study demonstrated a novel surface tension-based fabrication technique for the generation of millimeter-scale hydrogel beads using a liquid−liquid dual phase system. The “hydrogel millibeads” obtained by this method were larger than previously reported, highly uniform in shape and size with better ease of size control and a high degree of consistency and reproducibility between batches. In addition, human breast cancer cells were encapsulated within these hydrogel constructs to generate “tumor millibeads”, which were subsequently maintained in long-term 3D culture. Microscopic visualization using fluorescence imaging and microstructure analysis showed the morphology and uniform distribution of the cells within the 3D matrix and arrangement of cells with the surrounding scaffold material. Cell viability analysis revealed the creation of a core region of dead cells surrounded by healthy, viable cell layers at the periphery following long-term culture. These observations closely matched with those of native and in vivo tumors. Based on these results, this study established a rapidly reproducible surface tension-based fabrication technique for making spherical hydrogel millibeads and demonstrated the potential of this method in creating engineered 3D tumor tissues. It is envisioned that the developed hydrogel millibead system will facilitate the formation of physiologically relevant in vitro tumor models which will closely simulate the native tumor microenvironmental conditions and could enable future highthroughput testing of different anticancer drugs in preclinical trials.

1. INTRODUCTION Poly(ethylene glycol) (PEG)-based hydrogels have been extensively used for controlled delivery of drugs,1−3 macromolecules4,5 and nanoparticles,6,7 cell-delivery for tissue regeneration,8−10 and two-dimensional (2D) and three-dimensional (3D) culture of cells in vitro and in vivo.11−13 Some of the chief advantages of PEG for these applications are its high water content, ability to cross-link at physiological temperature and pH, covalent binding and controlled release of bioactive molecules,5 controlled proteolytic degradation of bioactive sites,14 tunable mechanical properties,15 and permeability to oxygen and nutrients.16,17 Numerous techniques have been developed to fabricate PEG-based hydrogels with controlled properties by both physical and chemical modifications.18−20 By developing additional techniques to control the specific size and geometry of PEG-based hydrogels, new applications and advances in current systems can be realized, such as engineering 3D tissues that better replicate healthy and diseased physiological microenvironments and for use in in vitro drug testing. © 2014 American Chemical Society

The suitability of PEG-based hydrogels in tissue-engineering applications has facilitated the development and fabrication of 3D in vitro models of cancer. These models aim to closely simulate the native tumor microenvironment and can be further used for high-throughput drug-screening processes. In previously established methods, in vitro 3D cancer models are usually formed by spherical, tightly packed aggregation of cancer cells (called tumor spheroids)21−23 or encapsulation of cancer cells within biomimetic materials and scaffolds of controlled size and geometry.13,24,25 Both techniques impose the desired limitations on oxygen and nutrient diffusion to the cancer cells, leading to hypoxia and cell death at the core of the aggregate and higher cellular viability and proliferation at the periphery. This phenomenon of hypoxic core formation is observed in native and in vivo tumors and has been widely Received: January 29, 2014 Revised: February 25, 2014 Published: March 11, 2014 3817

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Figure 1. Hydrogel millibeads formed by a dual-phase surface-tension based method. (A) Aqueous polymer precursor is pipetted onto the surface of the oil phase, where it remains suspended at the interface due to surface tension and is photo-cross-linked to yield hydrogel millibeads. Schematic not drawn to scale. (B) Polymer precursor floating at the air−oil interface prior to cross-linking. (C) Cross-linked hydrogel millibeads after light exposure. (D) Size comparison of hydrogel millibeads of three different pipetted volumes.

and the resulting tumor millibeads could effectively simulate larger, millimeter-scale tumors occurring in the human body. In addition, the hydrogel millibeads could also be used for the entrapment and controlled delivery of desired therapeutic agents at the site of delivery and thereby prove useful in the field of drug-delivery.

emulated in vitro as a means of increasing physiological relevance.26−28 A number of methods have been established to produce hydrogel microstructures using techniques including emulsification, photolithography, microfluidics, and micromolding.29−34 Specifically, PEG-based hydrogel microspheres have been widely investigated for various applications, with special focus placed on fabrication techniques to achieve desired specificity in size distribution.35−37 However, most of these methods are focused on creating relatively small hydrogel microparticles and microspheres (sub-500 μm scale) for efficient release of entrapped biomolecules or for ensuring good diffusion of nutrients and oxygen to maintain high viability of encapsulated cells.33,35 Our method reported here is the first to fabricate large-sized (millimeter-scale) PEG-based hydrogel millibeads for the 3D culture of encapsulated cancer cells, called “tumor millibeads”. This larger size is critical for replicating the native cancer microenvironment in vitro and thereby reproducing the cellular characteristics in an in vitro 3D context. In this study, we demonstrated a novel fabrication method of creating PEG-based hydrogel millibeads using an aqueoussuspension-in-oil method. By exploiting the differences in the surface tensions of air, aqueous, and oil phases, large, uniform, and size-controlled hydrogel millibeads were formed. Direct encapsulation of cancer cells in the hydrogel millibeads (tumor millibeads) and maintenance in 3D culture was demonstrated, followed by assessment of cell viability. This technique potentially facilitates the creation of a 3D tissue-engineered cancer model that could be used for the investigation of tumorigenic phenomena occurring in the cancer microenvironment in an in vitro setup. The fabricated millibeads were comparatively larger in size as compared to hydrogel microspheres obtained from other liquid−liquid emulsion systems,

2. EXPERIMENTAL SECTION All chemicals were obtained from Sigma-Aldrich (St. Louis, MO) unless mentioned otherwise. 2.1. PEGDA Synthesis. Poly(ethylene glycol) diacrylate (PEGDA) was prepared as described previously.38 Briefly, PEG (10kDA) was reacted with acryloyl chloride (1:4 molar ratio) in anhydrous dichloromethane with triethylamine (1:2 molar ratio) under argon overnight at 25 °C. The resulting PEGDA was purified by phase separation using 2 M K2CO3. The organic phase containing PEGDA was dried using anhydrous MgSO4 and filtered. Finally, PEGDA was precipitated in diethyl ether, filtered, and dried overnight under vacuum. The degree of acrylation was characterized by 1H NMR and the PEGDA was stored at −20 °C. 2.2. Cell Culture and Maintenance. MCF7 human breast adenocarcinoma cells were kindly provided by Dr. Richard C. Bird, College of Veterinary Medicine, Auburn University. MCF7 cells were cultured in DMEM (GIBCO, Carlsbad, CA) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Atlanta, GA), 1% (v/v) nonessential amino acids (NEAA) (Lonza, Walkersville, MD), 1% (v/ v) penicillin/streptomycin (GIBCO), 1% (v/v) Glutamax (GIBCO), and 1% (v/v) sodium pyruvate (GIBCO). The cells were maintained in tissue-culture flasks in a humidified atmosphere of 5% CO2 and constant temperature of 37 °C. Cell passages of 6−20 were used for all experiments. Cells cultured in 2D were enzymatically dissociated with 0.25% trypsin/2.21 mM EDTA (Corning Cellgro, Manassas, VA) and used for 3D encapsulation. 2.3. Millibead Formation. A dual photoinitiator, water-in-oil system was used for making hydrogel millibeads with and without cells.39 The aqueous phase hydrogel precursor solution was made by dissolving 10% (w/v) PEGDA in sterile PBS with 1.5% (v/v) 3818

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Figure 2. Cancer cells encapsulated within hydrogel millibeads. (A) Cells are mixed with the polymer precursor. The suspension is cross-linked to form tumor millibeads at the air−oil interface. The tumor millibeads are harvested, washed twice in DMEM to remove all oil, and cultured in media. Schematic not drawn to scale. Photomicrographs of cancer cells encapsulated within (B) 2 μL and (C) 5 μL tumor millibeads. The mean geometric diameter, D, was calculated from the major (a) and minor (b) axes of the fitted region of interest (ROI) around the millibeads:

triethanolamine (TEOA), 37 mM 1-vinyl-2-pyrrolidinone (NVP), and the aqueous phase photoinitiator, 0.1 mM Eosin Y in PBS. PEGDA precursor was cross-linked into hydrogels by free-radical visible light photopolymerization using Eosin Y as the primary photoinitiator, TEOA as the coinitiator, and (NVP) as the comonomer. To make the oil phase solution, 1.5% (v/v) triethanolamine and 3 μL/ml of oil phase photoinitiator (300 mg/mL Irgacure 651 (Ciba) in NVP) was mixed with mineral oil in a Petri dish. Microspheres were fabricated using a novel method: a set amount of precursor solution (2, 5, or 10 μL) was pulled up and then pipetted down slowly to form a droplet at the end of the pipet tip. Droplets were then carefully placed on the surface of the oil, so that the aqueous phase droplet remained floating at the oil-air interface. The floating droplets were cross-linked by exposure to light (365−700 nm wavelength) for 10 s (Figure 1). Upon completion of cross-linking, the newly formed hydrogel millibeads were removed from the oil phase, washed with PBS, and centrifuged at 200g for 2 min. To remove all residual oil, the wash and centrifugation step was repeated twice. The millibeads were then allowed to swell in PBS and reach equilibrium. 2.4. Cell Encapsulation within Millibeads. For cell encapsulation, trypsinized MCF7 cells were mixed with the aqueous phase hydrogel precursor solution at 60 × 106 cells/mL. Tumor millibeads were then formed according to the method described in the previous section. The millibeads containing cells were harvested, washed with DMEM, and incubated in cell culture media at 37 °C, 5% CO2 atmosphere for 5 days (Figure 2A) 2.5. Image Acquisition and Analysis. For each of the three different groups (2, 5, and 10 μL millibeads), two experimenters made 20 millibeads in 3 different batches (a total of 60 millibeads per condition). For each group, fabricated millibeads were pooled together and selected randomly for microscopic imaging and quantitative analysis. Phase contrast images of hydrogel millibeads with and without cells were acquired using an inverted Nikon Eclipse Ti microscope fitted with an Andor Luca S camera. For each millibead, the perimeter, P, the geometric mean diameter, D, and the projected area, A, were determined using ImageJ version 1.48a software (NIH). For each size group, 20 millibeads were analyzed. Data were exported to MS Excel, and a shape factor describing the sphericity of the millibeads (Φ) was calculated according to the method by Kelm et al.:40

P

(2)

a×b

The spherical volume of the millibeads, based on the projected area, was calculated as follows: V=

4π ⎛⎜ D ⎞⎟3 3 ⎝2⎠

(3)

The shape factor corrected volume, V′, is given by: (4)

V ′ = ΦV

2.6. Hydrogel Swelling. To assess hydrogel millibead swelling, size and percentage increase of volume were evaluated with time. Acellular hydrogel millibeads were allowed to swell in PBS and images of swollen hydrogels were acquired at 5, 30, 60, and 120 min after incubation in PBS. The images were analyzed using ImageJ to determine geometric mean diameter, volume, and sphericity of swollen millibeads as described in the previous section. For each size group 30 millibeads were analyzed. The percentage increase in volume of hydrogel millibeads due to swelling was calculated using the following formula:

% increase in volume =

final volume − initial volume × 100 final volume

(5) 2.7. Hydrogel Diffusion. The rate of diffusion of entrapped moieties within the hydrogel was analyzed. Hydrogel millibeads entrapped with 5 mg/mL of TRITC−dextran, average molecular weight 4400 Da, were formed as per the previous protocol. After removal of residual oil, the millibeads were allowed to incubate in 50 μL of PBS (for each millibead) to ensure that the entire millibead is completely submerged and to facilitate the diffusion of the entrapped TRITC−dextran from within the hydrogel matrix to the surrounding PBS. The PBS was collected at specific time points in a 96-well plate, and 50 μL of fresh PBS was immediately added to the hydrogel millibeads. The collected PBS containing the TRITC−dextran was analyzed through a plate reader at 540/25 excitation wavelength and 590/35 emission wavelength. Known concentrations of TRITC− dextran in PBS were used for the standard curve, and fresh PBS without TRITC−dextran was used as the blank. This procedure was continued until there was no detectable signal in the collected PBS.

4A π

π× Φ=

D=

(1) 3819

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The absolute fluorescence intensity obtained from the plate reader was converted into concentration based on the standard curve and was reported as the percentage of the total TRITC−dextran that diffused out over the entire period of observation. For each size group, 10 samples were analyzed for each time point. 2.8. SEM Imaging. Hydrogel millibeads with and without cells were visualized through scanning electron microscopy (SEM). First, the samples were fixed in 3% glutaraldehyde for 2 h at room temperature and then postfixated in 2% osmium tetroxide for 2 h, each step being followed by two PBS washes. The fixed samples were dehydrated in graded ethanol series sequentially and made to undergo chemical drying by hexamethyldisilazane (HMDS) for 30 min, followed by air drying. Finally, the dry samples were sputter-coated with gold (EMS 550X Auto Sputter Coating Device), mounted on aluminum stubs and imaged using SEM (Zeiss EVO 50 SEM). The average size of cells encapsulated within tumor millibeads was also evaluated from the obtained micrographs using ImageJ. 2.9. Fluorescence Staining and Analysis. Cancer cells were encapsulated within hydrogel millibeads and cultured in media for either 3 h or 5 days. They were subsequently washed with PBS and incubated in the Live/Dead Cell viability stain (Invitrogen, Carlsbad, CA) for 1 h. The samples were washed with PBS again and imaged under an inverted Nikon Ti microscope. Acquired images were further analyzed using NIS Elements Viewer and ImageJ software. Cell encapsulated millibeads were washed with PBS to remove all media and fixed with 4% paraformaldehyde for 1 h at room temperature. They were subsequently washed and permeabilized with 0.5% Triton X-100 for 15 min. The millibeads were blocked with blocking buffer (2% bovine serum albumin + 5% fetal calf serum in PBS (GIBCO)) for 3 h. They were subsequently stained with Alexa Fluor 488 Phalloidin and DAPI (Invitrogen) in blocking buffer overnight. The millibeads were washed in PBS and dehydrated using graded ethanol series. After air-drying, they were mounted on coverslips and imaged using confocal microscopy to observe encapsulated cell morphology and distribution (Nikon AI Confocal Scanning Laser Microscope). 2.10. Statistical Analysis. All statistical analysis was performed using Minitab 16 Statistical Software (Minitab Inc.). One-way analysis of variance (ANOVA) with Tukey’s family error rate of 5% was used to evaluate statistical significance between multiple groups. An assumption of equal variances between groups was made, based on the large, equal, and independent sample size for each group, low variance ratio (