Dual-Phase, Surface Tension-Based Fabrication Method for Generation of Tumor Millibeads

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 high-throughput testing of different anticancer drugs in preclinical trials.

PEG‐fibrinogen hydrogels for three‐dimensional breast cancer cell culture

“…However, cells located in the interior regions of the tumor tissue experience diffusional gradients of oxygen (leading to hypoxia) and nutrients (leading to accumulation of waste metabolites and acidic pH) . Previously, we have shown that MCF7 cells cultured within spheroidal PEGDA hydrogel millibeads (2 mm diameter) exhibit viable cell layers in the hydrogel periphery, but undergo significant cell death in the interior regions of the millibeads . In another study, cancer cells encapsulated within transglutaminase‐crosslinked gelatin hydrogels also displayed growth and morphological heterogeneity due to onset of hypoxia in the central regions of millimeter‐scale large hydrogel constructs .…”

“…56,57 Previously, we have encapsulated MCF7 cells within PEGDA tumor millibeads (2 mm in diameter) at 60 3 10 6 cells/mL and observed the formation of a central region of dead cells and a peripheral rim of viable cells, representative of the necrotic core of native tumors. 11 In our experience, high encapsulation densities and dense cell colonies at the hydrogel edge are necessary to establish a mass transfer gradient from the hydrogel periphery to the hydrogel interior for constructs of these dimensions and thereby demonstrate locational tumor heterogeneity as described in Figure 10.…”

“…44,62 Previously, we have shown that MCF7 cells cultured within spheroidal PEGDA hydrogel millibeads (2 mm diameter) exhibit viable cell layers in the hydrogel periphery, but undergo significant cell death in the interior regions of the millibeads. 11 In another study, cancer cells encapsulated within transglutaminase-crosslinked gelatin hydrogels also displayed growth and morphological heterogeneity due to onset of hypoxia in the central regions of millimeter-scale large hydrogel constructs. 63 Since a distinct subpopulation of MCF7, SK-BR-3, and MDA-MB-231 cells in the PF-based tumor constructs appear to be either nonproliferative or morphologically noninvasive, these cells could be used in future investigations of tumor dormancy or cancer stem cell-like characteristics.…”

“…PEGDA was prepared as described previously. 11 Briefly, poly(ethylene glycol) (Molecular weight: 10 kDA) was reacted with acryloyl chloride (molar ratio of 1:4) in anhydrous dichloromethane with triethylamine (molar ratio of 1:2) under argon overnight at 258C. The PEGDA obtained from the reaction was purified by phase separation using 1.5 M K 2 CO 3 .…”

“…5,6 To bridge this gap in correlation, bioengineered cancer models have been developed by culturing cancer cells within 3D biomimetic scaffolds that mimic key aspects of the native tumor microenvironment under in vitro conditions. [6][7][8] These scaffolds used for simulating native extracellular matrix (ECM) characteristics are usually obtained from natural (e.g., collagen, Matrigel, agarose), 3,4,9,10 synthetic [e.g., poly(ethylene glycol), poly(lactic-co-glycolic acid)], [11][12][13] or hybrid (combination of natural and synthetic) sources. 14,15 One of the components of the native tumor ECM is fibrinogen, which is known to be secreted and deposited by cancer cells.…”