Jessie S. Jeon

Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels

This protocol describes a simple but robust microfluidic assay combining three-dimensional (3D) and two-dimensional (2D) cell culture. The microfluidic platform comprises hydrogel-incorporating chambers between surface-accessible microchannels. By using this platform, well-defined biochemical and biophysical stimuli can be applied to multiple cell types interacting over distances of <1 mm, thereby replicating many aspects of the in vivo microenvironment. Capabilities exist for time-dependent manipulation of flow and concentration gradients as well as high-resolution real-time imaging for observing spatial-temporal single-cell behavior, cell-cell communication, cell-matrix interactions and cell population dynamics. These heterotypic cell type assays can be used to study cell survival, proliferation, migration, morphogenesis and differentiation under controlled conditions. Applications include the study of previously unexplored cellular interactions, and they have already provided new insights into how biochemical and biophysical factors regulate interactions between populations of different cell types. It takes 3 d to fabricate the system and experiments can run for up to several weeks.

Human 3D vascularized organotypic microfluidic assays to study breast cancer cell extravasation

Significance The cancer biology seed-and-soil paradigm recognizes the existence of organ-specific patterns of metastasization that drive the spread of selected primary tumors toward specific secondary loci. However, despite efforts to model the organotypic microenvironment, the organ specificity of cancer metastases needs to be elucidated. The relevance of this study lies in the generation of a human vascularized organ-specific microenvironment, which can be used to investigate and tune the extravasation process of metastatic tumor cells. Furthermore, beyond mimicking the pro- or antimetastatic signatures of different microenvironments, our microfluidic model provides insights into different properties of organ-specific endothelia. This study paves the way toward advanced in vitro models to screen for highly tailored organ-specific therapeutics and investigate key molecular pathways involved in organ-specific metastases. A key aspect of cancer metastases is the tendency for specific cancer cells to home to defined subsets of secondary organs. Despite these known tendencies, the underlying mechanisms remain poorly understood. Here we develop a microfluidic 3D in vitro model to analyze organ-specific human breast cancer cell extravasation into bone- and muscle-mimicking microenvironments through a microvascular network concentrically wrapped with mural cells. Extravasation rates and microvasculature permeabilities were significantly different in the bone-mimicking microenvironment compared with unconditioned or myoblast containing matrices. Blocking breast cancer cell A3 adenosine receptors resulted in higher extravasation rates of cancer cells into the myoblast-containing matrices compared with untreated cells, suggesting a role for adenosine in reducing extravasation. These results demonstrate the efficacy of our model as a drug screening platform and a promising tool to investigate specific molecular pathways involved in cancer biology, with potential applications to personalized medicine.

Mechanisms of tumor cell extravasation in an in vitro microvascular network platform

A deeper understanding of the mechanisms of tumor cell extravasation is essential in creating therapies that target this crucial step in cancer metastasis. Here, we use a microfluidic platform to study tumor cell extravasation from in vitro microvascular networks formed via vasculogenesis. We demonstrate tight endothelial cell-cell junctions, basement membrane deposition and physiological values of vessel permeability. Employing our assay, we demonstrate impaired endothelial barrier function and increased extravasation efficiency with inflammatory cytokine stimulation, as well as positive correlations between the metastatic potentials of MDA-MB-231, HT-1080, MCF-10A and their extravasation capabilities. High-resolution time-lapse microscopy reveals the highly dynamic nature of extravasation events, beginning with thin tumor cell protrusions across the endothelium followed by extrusion of the remainder of the cell body through the formation of small (~1 μm) openings in the endothelial barrier which grows in size (~8 μm) to allow for nuclear transmigration. No disruption to endothelial cell-cell junctions is discernible at 60×, or by changes in local barrier function after completion of transmigration. Tumor transendothelial migration efficiency is significantly higher in trapped cells compared to non-trapped adhered cells, and in cell clusters versus single tumor cells.

In Vitro Model of Tumor Cell Extravasation

Tumor cells that disseminate from the primary tumor and survive the vascular system can eventually extravasate across the endothelium to metastasize at a secondary site. In this study, we developed a microfluidic system to mimic tumor cell extravasation where cancer cells can transmigrate across an endothelial monolayer into a hydrogel that models the extracellular space. The experimental protocol is optimized to ensure the formation of an intact endothelium prior to the introduction of tumor cells and also to observe tumor cell extravasation by having a suitable tumor seeding density. Extravasation is observed for 38.8% of the tumor cells in contact with the endothelium within 1 day after their introduction. Permeability of the EC monolayer as measured by the diffusion of fluorescently-labeled dextran across the monolayer increased 3.8 fold 24 hours after introducing tumor cells, suggesting that the presence of tumor cells increases endothelial permeability. The percent of tumor cells extravasated remained nearly constant from1 to 3 days after tumor seeding, indicating extravasation in our system generally occurs within the first 24 hours of tumor cell contact with the endothelium.

In vitro 3D collective sprouting angiogenesis under orchestrated ANG-1 and VEGF gradients

Sprouting angiogenesis requires a coordinated guidance from a variety of angiogenic factors. Here, we have developed a unique hydrogel incorporating microfluidic platform which mimics the physiological microenvironment in 3D under a precisely orchestrated gradient of soluble angiogenic factors, VEGF and ANG-1. The system enables the quantified investigation in chemotactic response of endothelial cells during the collective angiogenic sprouting process. While the presence of a VEGF gradient alone was sufficient in inducing a greater number of tip cells, addition of ANG-1 to the VEGF gradient enhanced the number of tip cells that are attached to collectively migrated stalk cells. The chemotactic response of tip cells attracted by the VEGF gradient and the stabilizing role of ANG-1 were morphologically investigated, elucidating the 3D co-operative migration of tip and stalk cells as well as their structures. We found that ANG-1 enhanced the connection of the stalk cells with the tip cells, and then the direct connection regulated the morphogenesis and/or life cycle of stalk cells.

Generation of 3D functional microvascular networks with human mesenchymal stem cells in microfluidic systems

The generation of functional microvascular networks is critical for the development of advanced in vitro models to replicate pathophysiological conditions. Mural cells provide structural support to blood vessels and secrete biomolecules contributing to vessel stability and functionality. We investigated the role played by two endothelium-related molecules, angiopoietin (Ang-1) and transforming growth factor (TGF-β1), on bone marrow-derived human mesenchymal stem cell (BM-hMSC) phenotypic transition toward a mural cell lineage, both in monoculture and in direct contact with human endothelial cells (ECs), within 3D fibrin gels in microfluidic devices. We demonstrated that the effect of these molecules is dependent on direct heterotypic cell-cell contact. Moreover, we found a significant increase in the amount of α-smooth muscle actin in microvascular networks with added VEGF and TGF-β1 or VEGF and Ang-1 compared to networks with added VEGF alone. However, the addition of TGF-β1 generated a non-interconnected microvasculature, while Ang-1 promoted functional networks, confirmed by microsphere perfusion and permeability measurements. The presence of mural cell-like BM-hMSCs coupled with the addition of Ang-1 increased the number of network branches and reduced mean vessel diameter compared to EC only vasculature. This system has promising applications in the development of advanced in vitro models to study complex biological phenomena involving functional and perfusable microvascular networks.

Hot embossing for fabrication of a microfluidic 3D cell culture platform

Clinically relevant studies of cell function in vitro require a physiologically-representative microenvironment possessing aspects such as a 3D extracellular matrix (ECM) and controlled biochemical and biophysical parameters. A polydimethylsiloxane (PDMS) microfluidic system with a 3D collagen gel has previously served for analysis of factors inducing different responses of cells in a 3D microenvironment under controlled biochemical and biophysical parameters. In the present study, applying the known commercially-viable manufacturing methods to a cyclic olefin copolymer (COC) material resulted in a microfluidic device with enhanced 3D gel capabilities, controlled surface properties, and improved potential to serve high-volume applications. Hot embossing and roller lamination molded and sealed the microfluidic device. A combination of oxygen plasma and thermal treatments enhanced the sealing, ensured proper placement of the 3D gel, and created controlled and stable surface properties within the device. Culture of cells in the new device indicated no adverse effects of the COC material or processing as compared to previous PDMS devices. The results demonstrate a methodology to transition microfludic devices for 3D cell culture from scientific research to high-volume applications with broad clinical impact.

A quantitative microfluidic angiogenesis screen for studying anti-angiogenic therapeutic drugs

Anti-angiogenic therapy, which suppresses tumor growth by disrupting oxygen and nutrient supply from blood to the tumor, is now widely accepted as a treatment for cancer. To investigate the mechanisms of action of these anti-angiogenesis drugs, new three dimensional (3D) cell culture-based drug screening models are increasingly employed. However, there is no in vitro high-throughput screening (HTS) angiogenesis assay that can provide uniform culture conditions for the quantitative assessment of physiological responses to chemoattractant reagents under various concentrations of anti-angiogenesis drugs. Here we describe a method for screening and quantifying the vascular endothelial growth factor (VEGF)-induced chemotactic response on human umbilical vein endothelial cells (HUVECs) cultured with different concentrations of bortezomib, a selective 26S proteasome inhibitor. With this quantitative microfluidic angiogenesis screen (QMAS), we demonstrate that bortezomib-induced endothelial cell death is preceded by a series of morphological changes that develop over several days. We also explore the mechanisms by which bortezomib can inhibit angiogenesis.

In vitro models of the metastatic cascade: from local invasion to extravasation.

A crucial event in the metastatic cascade is the extravasation of circulating cancer cells from blood capillaries to the surrounding tissues. The past 5 years have been characterized by a significant evolution in the development of in vitro extravasation models, which moved from traditional transmigration chambers to more sophisticated microfluidic devices, enabling the study of complex cell-cell and cell-matrix interactions in multicellular, controlled environments. These advanced assays could be applied to screen easily and rapidly a broad spectrum of molecules inhibiting cancer cell endothelial adhesion and extravasation, thus contributing to the design of more focused in vivo tests.

Vasculature-On-A-Chip for In Vitro Disease Models

Vascularization, the formation of new blood vessels, is an essential biological process. As the vasculature is involved in various fundamental physiological phenomena and closely related to several human diseases, it is imperative that substantial research is conducted on characterizing the vasculature and its related diseases. A significant evolution has been made to describe the vascularization process so that in vitro recapitulation of vascularization is possible. The current microfluidic systems allow elaborative research on the effects of various cues for vascularization, and furthermore, in vitro technologies have a great potential for being applied to the vascular disease models for studying pathological events and developing drug screening platforms. Here, we review methods of fabrication for microfluidic assays and inducing factors for vascularization. We also discuss applications using engineered vasculature such as in vitro vascular disease models, vasculature in organ-on-chips and drug screening platforms.