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Abstract The mammary gland is a highly vascularized organ influenced by sex hormones including estrogen (E2) and progesterone (P4). Beyond whole‐organism studies in rodents or cell monocultures, hormonal effects on the breast microvasculature remain largely understudied. Recent methods to generate 3D microvessels on‐chip have enabled direct observation of complex vascular processes; however, these models often use non‐tissue‐specific cell types, such as human umbilical vein endothelial cells (HUVECs) and fibroblasts from various sources. Here, novel mammary‐specific microvessels are generated by coculturing primary breast endothelial cells and fibroblasts under optimized culture conditions. These microvessels are mechanosensitive (to interstitial flow) and require endothelial–stromal interactions to develop fully perfusable vessels. These mammary‐specific microvessels are also responsive to exogenous stimulation by sex hormones. When treated with combined E2 and P4, corresponding to the four phases of the menstrual cycle (period, follicular, ovular, and luteal), vascular remodeling and barrier function are altered in a phase‐dependent manner. The presence of high E2 (ovulation) promotes vascular growth and remodeling, corresponding to high depletion of proangiogenic factors, whereas high P4 concentrations (luteal) promote vascular regression. The effects of combined E2 and P4 hormones are not only dose‐dependent but also tissue‐specific, as are shown by similarly treating non‐tissue‐specific HUVEC microvessels.

Abstract Optical microscopy techniques are a popular choice for visualizing micro-agents. They generate images with relatively high spatiotemporal resolution but do not reveal encoded information for distinguishing micro-agents and surroundings. This study presents multicolor fluorescence microscopy for rendering color-coded identification of mobile micro-agents and dynamic surroundings by spectral unmixing. We report multicolor microscopy performance by visualizing the attachment of single and cluster micro-agents to cancer spheroids formed with HeLa cells as a proof-of-concept for targeted drug delivery demonstration. A microfluidic chip is developed to immobilize a single spheroid for the attachment, provide a stable environment for multicolor microscopy, and create a 3D tumor model. In order to confirm that multicolor microscopy is able to visualize micro-agents in vascularized environments, in vitro vasculature network formed with endothelial cells and ex ovo chicken chorioallantoic membrane are employed as experimental models. Full visualization of our models is achieved by sequential excitation of the fluorophores in a round-robin manner and synchronous individual image acquisition from three-different spectrum bands. We experimentally demonstrate that multicolor microscopy spectrally decomposes micro-agents, organic bodies (cancer spheroids and vasculatures), and surrounding media utilizing fluorophores with well-separated spectrum characteristics and allows image acquisition with 1280 $$\times$$ × 1024 pixels up to 15 frames per second. Our results display that real-time multicolor microscopy provides increased understanding by color-coded visualization regarding the tracking of micro-agents, morphology of organic bodies, and clear distinction of surrounding media.

Given the dynamic nature of engineered vascular networks within biofabricated tissue analogues, it is instrumental to have control over the constantly evolving biochemical cues within synthetic matrices throughout tissue remodeling. Incorporation of pro-angiogenic vascular endothelial growth factor (VEGF165) specific aptamers into cell-instructive polymer networks is shown to be pivotal for spatiotemporally controlling the local bioactivity of VEGF that selectively elicit specific cell responses. To harness this effect and quantitatively unravel its spatial resolution, herein, bicomponent micropatterns consisting of VEGF165 specific aptamer-functionalized gelatin methacryloyl (GelMA) (aptamer regions) overlaid with pristine GelMA regions using visible-light photoinitiators (Ru/SPS) were fabricated via two-step photopatterning approach. For the 3D co-culture study, human umbilical vein-derived endothelial cells and mesenchymal stromal cells were used as model cell types. Bicomponent micropatterns with spatially defined spacings (300/500/800 ​μm) displayed high aptamer retention, aptamer-fluorescent complementary sequence (CSF) molecular recognition and VEGF sequestration localized within patterned aptamer regions. Stiffness gradient at the interface of aptamer and GelMA regions was observed with high modulus in aptamer region followed by low stiffness GelMA regions. Leveraging aptamer-tethered VEGF's dynamic affinity interactions with CS that upon hybridization facilitates triggered VEGF release, co-culture studies revealed unique characteristics of aptamer-tethered VEGF to form spatially defined luminal vascular networks covered with filopodia-like structures in vitro (spatial control) and highlights their ability to control network properties including orientation over time using CS as an external trigger (temporal control). Moreover, the comparison of single and double exposed regions within micropatterns revealed differences in cell behavior among both regions. Specifically, the localized aptamer-tethered VEGF within single exposed aptamer regions exhibited higher cellular alignment within the micropatterns till d5 of culture. Taken together, this study highlights the potential of photopatterned aptamer-tethered VEGF to spatiotemporally regulate vascular morphogenesis as a tool for controlling vascular remodeling in situ.

Abstract Fluid flow shear stresses are strong regulators for directing the organization of vascular networks. Knowledge of structural and flow dynamics information within complex vasculature is essential for tuning the vascular organization within engineered tissues, by manipulating flows. However, reported investigations of vascular organization and their associated flow dynamics within complex vasculature over time are limited, due to limitations in the available physiological pre-clinical models, and the optical inaccessibility and aseptic nature of these models. Here, we developed laser speckle contrast imaging (LSCI) and side-stream dark field microscopy (SDF) systems to map the vascular organization, spatio-temporal blood flow fluctuations as well as erythrocytes movements within individual blood vessels of developing chick embryo, cultured within an artificial eggshell system. By combining imaging data and computational simulations, we estimated fluid flow shear stresses within multiscale vasculature of varying complexity. Furthermore, we demonstrated the LSCI compatibility with bioengineered perfusable muscle tissue constructs, fabricated via molding techniques. The presented application of LSCI and SDF on perfusable tissues enables us to study the flow perfusion effects in a non-invasive fashion. The gained knowledge can help to use fluid perfusion in order to tune and control multiscale vascular organization within engineered tissues.