Three-dimensional microfluidic collagen hydrogels for investigating flow-mediated tumor-endothelial signaling and vascular organization

Abstract

Hyperpermeable tumor vessels are responsible for elevated interstitial fluid pressure and altered flow patterns within the tumor microenvironment. These aberrant hydrodynamic stresses may enhance tumor development by stimulating the angiogenic activity of endothelial cells lining the tumor vasculature. However, it is currently not known to what extent shear forces affect endothelial organization or paracrine signaling during tumor angiogenesis. The objective of this study was to develop a three-dimensional (3D), in vitro microfluidic tumor vascular model for coculture of tumor and endothelial cells under varying flow shear stress conditions. A central microchannel embedded within a collagen hydrogel functions as a single neovessel through which tumor-relevant hydrodynamic stresses are introduced and quantified using microparticle image velocimetry (μ-PIV). This is the first use of μ-PIV in a tumor representative, 3D collagen matrix comprised of cylindrical microchannels, rather than planar geometries, to experimentally measure flow velocity and shear stress. Results demonstrate that endothelial cells develop a confluent endothelium on the microchannel lumen that maintains integrity under physiological flow shear stresses. Furthermore, this system provides downstream molecular analysis capability, as demonstrated by quantitative RT-PCR, in which, tumor cells significantly increase expression of proangiogenic genes in response to coculture with endothelial cells under low flow conditions. This work demonstrates that the microfluidic in vitro cell culture model can withstand a range of physiological flow rates and permit quantitative measurement of wall shear stress at the fluid-collagen interface using μ-PIV optical flow diagnostics, ultimately serving as a versatile platform for elucidating the role of fluid forces on tumor-endothelial cross talk.

FIG. 1.

Tumor-endothelial coculture in microfluidic collagen hydrogels. (a) Schematic of a three-dimensional (3D) microfluidic tumor vascular model in which cancer cells seeded in the bulk of hydrogel surround endothelial cells lining the lumenal surface of the central microchannel. (b) Coculture maintains growth of MDA-MB-231-GFP breast cancer cells and telomerase immortalized microvascular endothelial (TIME)-red fluorescent protein (RFP) cells within physiologically relevant geometries. Image 24 h postculture. Scale bar 200 μm. Color images available online at www.liebertpub.com/tec

FIG. 2.

Fabrication of microfluidic collagen hydrogels and perfusion setup. (a) Fluorinated ethylene propylene tubing fit concentrically with a 2.0′′ 22G needle and capped with polydimethylsiloxane sleeves. (b) Microfluidic collagen hydrogel after polymerization and removal of the needle. (c) Magnified view of the central microchannel. (d) Introduction of flow into the microchannel using a syringe pump and in-line bubble trap. (e) Close-up view of 0.5′′ 22G needles inserted into either end of the microchannel for dynamic culture. Color images available online at www.liebertpub.com/tec

FIG. 3.

Shear stress preconditioning protocol to develop a confluent endothelium. (a) A graded increase in shear stress (τ_W=0.01 to 0.1 dyn/cm²) over 72 h best encourages the development of a confluent endothelium, after which, a rate of increase in shear of ∼5.5 (dyn/cm²)(dyn/cm²)h⁻¹ was used to reach a target wall shear stress (WSS) of τ_W=1, 4, or 10 dyn/cm² in t=9.71, 42.12, or 106.58 min, respectively, and maintained for a total of 6 h. (b) TIME-RFP cells exhibit progressive adhesion, proliferation, and elongation in the direction of flow during the 72-h preconditioning scheme. Color images available online at www.liebertpub.com/tec

FIG. 4.

Schematic of microparticle image velocimetry (μ-PIV) flow diagnostics μ-PIV, a noninvasive flow measurement technique with a high spatial and temporal resolution, was used to measure velocities in the collagen microchannel. (a) The microfluidic collagen scaffold interfaced with the u-PIV imaging system (adapted from Fouras et al.). (b) Fluorescent tracer particles in the flow are illuminated and imaged by a high-speed camera. (c) Cross correlation of image pairs is used to calculate particle displacement, which, with known image frequency, quantifies the velocity field. Color images available online at www.liebertpub.com/tec

FIG. 5.

TIME cells develop a confluent endothelium and maintain integrity after exposure to each target WSS (τ_W). The endothelium with fluorescently labeled F-Actin and DAPI after the 72-h preconditioning scheme and plus an additional 6-h exposure of τ_W=1, 4, or 10 dyn/cm² demonstrate increased alignment in the direction of flow as a function of increasing WSS. Scale bar 50 μm. Color images available online at www.liebertpub.com/tec

FIG. 6.

Tumor cell viability and proliferation in the collagen hydrogel. (a) MDA-MB-231 cells remain>90% viable and (b) demonstrate increased proliferation within the collagen matrix for the duration of the 3-day experiment. Data expressed as average viability±standard deviation (n=3). Color images available online at www.liebertpub.com/tec

FIG. 7.

Experimentally measured velocity profiles and WSS in the microchannel. (a) Parabolic velocity profiles for each target WSS calculated from experimental PIV measurements (red) and compared to the Poiseuille solution (black). Data shown in endothelialized microchannels. (b) μ-PIV measured in both acellular and endothelialized microchannels for each target WSS and compared with theoretical WSS. WSS calculated based on the linear portion of the measured velocity gradient and measured viscosity of culture media (0.78 cP). Data expressed as nondimensionalized WSS (τ/ρv2)±standard deviation. Color images available online at www.liebertpub.com/tec

FIG. 8.

Three-dimensional, dynamic coculture with endothelial cells significantly increases tumor-expressed angiogenic genes. After 24 h, MDA-MB-231 cells significantly upregulate matrix metalloproteinase 9 (MMP9), platelet-derived growth factor B (PDGFB), and angiopoietin 2 (ANGPT2) under 3D static conditions relative to 2D tissue culture-treated polystyrene controls. The introduction of a low flow rate (50 μL/min) further increased PDGFB as well as significantly increased vascular endothelial growth factor A (VEGFA) gene expression relative to 3D static conditions. The presence of endothelial cells without flow had a significant effect on tumor expressed VEGFA; however, in the presence of flow, all tumor-expressed proangiogenic genes were significantly upregulated during coculture, relative to MDA-MB-231 monocultures under 3D dynamic conditions. Relative mRNA to GAPDH mRNA expressed as a fold induction±standard deviation (n=4). *p<0.05.