Measurement of cell metabolism in moderate-throughput to high-throughput organ-on-chip (OOC) systems would expand the range of data collected for studying drug effects or disease in physiologically relevant tissue models. However, current measurement approaches rely on fluorescent imaging or colorimetric assays that are focused on endpoints, require labels or added substrates, and lack real-time data. Here, we integrated optical-based oxygen sensors in a high-throughput OOC platform and developed an approach for monitoring cell metabolic activity in an array of membrane bilayer devices. Each membrane bilayer device supported a culture of human renal proximal tubule epithelial cells on a porous membrane suspended between two microchannels and exposed to controlled, unidirectional perfusion and physiologically relevant shear stress for several days. For the first time, we measured changes in oxygen in a membrane bilayer format and used a finite element analysis model to estimate cell oxygen consumption rates (OCRs), allowing comparison with OCRs from other cell culture systems. Finally, we demonstrated label-free detection of metabolic shifts in human renal proximal tubule cells following exposure to FCCP, a drug known for increasing cell oxygen consumption, as well as oligomycin and antimycin A, drugs known for decreasing cell oxygen consumption. The capability to measure cell OCRs and detect metabolic shifts in an array of membrane bilayer devices contained within an industry standard microtiter plate format will be valuable for analyzing flow-responsive and physiologically complex tissues during drug development and disease research.
Abstract Cancer cell metastases arise due to a series of biological processes starting from malignant tumor cells invading from primary to distal organs. Bone is one of the preferred sites of metastasis for various tumors including breast cancer. Understanding metastatic invasion to the secondary site requires studying the individual steps in metastasis, particularly those leading up to and immediately following extravasation of a circulating tumor cell from a blood vessel into the remote tissue. Microfluidic systems can provide cells with a controllable and reproducible 3D bone micro-environment to study cancer extravasation to a degree not possible by standard tissue culture methods while also simultaneously providing in situ imaging capabilities for visualization. We investigate the critical steps of cancer extravasation: tumor cells adhering to the endothelium and subsequently transmigrate across it into a hydrogel model for the bone extracellular space. Our model system consists of a functional microvascular network generated through vasculogenesis in a bone-mimicking microenvironment all within a microfluidic system. The microfluidic system contains a central hydrogel region flanked by two lateral media channels. The hydrogel region is filled with cells dispersed inside a fibrin gel. The two channel system enables easy access to the microvascular network for the addition of cancer cells for extravasation. Cells of passage 6 or lower MSCs, harvested from patients undergoing hip arthroplasty, were differentiated to osteo lineage. GFP-human umbilical vein endothelial cells (HUVECs) were suspended at 2·107 cells/ml in EMG2 + thrombin and combined with osteo-differentiated and non-differentiated MSCs mixed at a 9:1 ratio, and inserted into the gel channel to complete the seeding. RFP-expressing BOKL, which is an MDA-MB-231 cell line specific to bone metastasis enabled live-cell imaging via fluorescent microscopy. We created a functional microvascular network in a bone cell-conditioned microenvironment with the addition of osteo-differentiated and non-differentiated MSCs. Immunofluorescent imaging of α-smooth muscle actin in the system confirmed the presence of mural cells differentiated from MSCs wrapped around the generated vascular network. Visual confirmation of 3μm microspheres flowing through the vessels confirmed that the vascular network generated in this system was indeed perfusable. Finally, the osteo-cell conditioned microenvironment was confirmed by staining for osteocalcin. BOKL were then perfused into the microvasculature to model the extravasation process. The percentage of cancer cell extravasation in a device containing a microvascular network seeded with osteo-differentiated MSCs was 3.8 fold higher (56.5±4.8%) than the case with HUVECs alone (14.7±3.7%). Permeability values of the vasculature were analyzed by monitoring the leakage of fluorescent 70kDa dextran from the microvessels over time. Vasculature in the bone cell-conditioned microenvironment exhibited a higher permeability (4.12±0.75)·10-6 cm/s compared to the HUVEC only condition (0.89±0.31)·10-6 cm/s. While the increase in extravasation rates may due to a combination of factors, the higher permeability of the vasculature in osteo-cell conditioned environment may also be one of the contributing factors. Microfluidics offers the capability to create organ-specific microenvironments that can capture extravasation events, leading to tumor metastasis. This system therefore captures certain aspects of cell-cell and cell-matrix interactions for preferential metastasis to bone, offering potential for drug screening. Citation Format: Jessie S. Jeon, Simone Bersini, Gabriele Dubini, Joseph Charest, Matteo Moretti, Roger D. Kamm. Extravasation of breast cancer cells to a bone-cell conditioned microenvironment in functional 3D microvascular networks generated by vasculogenesis in a microfluidic system. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B23. doi:10.1158/1538-7445.CHTME14-B23
The intestinal epithelium comprises diverse cell types and executes many specialized functions as the primary interface between luminal contents and internal organs. A key function provided by the epithelium is maintenance of a barrier that protects the individual from pathogens, irritating luminal contents, and the microbiota. Disruption of this barrier can lead to inflammatory disease within the intestinal mucosa, and, in more severe cases, to sepsis. Animal models to study intestinal permeability are costly and not entirely predictive of human biology. Here we present a model of human colon barrier function that integrates primary human colon stem cells into Draper's PREDICT96 microfluidic organ-on-chip platform to yield a high-throughput system appropriate to predict damage and healing of the human colon epithelial barrier. We have demonstrated pharmacologically induced barrier damage measured by both a high throughput molecular permeability assay and transepithelial resistance. Using these assays, we developed an Inflammatory Bowel Disease-relevant model through cytokine induced damage that can support studies of disease mechanisms and putative therapeutics.
A combination of nanoimprint lithography and microcontact printing was used to create cell substrates with well-defined nanotopographic patterns of grooves overlaid with independently controlled micropatterned chemical domains. Qualitative analysis of osteoblast-like cells cultured on the substrates showed alignment of cells and cell features to the nanotopographic grooves when surface chemistry was either uniform or a pattern of dots. When surface chemistry on the substrate was a pattern of lanes, cells aligned to the lanes. On all substrates, small cellular extensions, or filopodia, displayed no particular alignment to either nanotopographic grooves or chemical patterns. Large cell extensions were observed only parallel to either nanotopographic grooves or chemical lanes. The techniques used provide an easily scaleable approach to creating cell substrates that will aid in studying the relative impact and interplay of chemical patterns and mechanical topography on cellular responses.
This work investigates processing parameters affecting replicated feature size during hot embossing micro-manufacturing. Silicon micromachined masters were heated and pressed into polymer layers of different thermophysical properties. Imprinting with loads ranging from 20–35 MPa, load rates from 1–15 MPa/sec, load times from 90–115 sec, and imprint temperatures at, below, and above the polymer glass transition temperature (Tg) replicated features in polymer with varying degrees of conformity. Replicated features were measured by profilometry and inspected by scanning electron microscopy, revealing polymer feature heights ranging from 25–100% conformal matching of silicon master features and polymer feature widths closely matching the period of features on the silicon master. Statistical analysis determined replicated feature height was positively dependent on tip sharpness, master feature height, temperature, and load rate while negatively dependent on master feature width. Replicated feature width was found to depend positively on master feature height and width, temperature, load rate, and load time. Optimization of imprint parameters during hot embossing micro/nano-manufacturing can possibly lead to a high-throughput manufacturing process offering nanometer resolution.
Models of reabsorptive barriers require both a means to provide realistic physiologic cues to and quantify transport across a layer of cells forming the barrier. Here we have topographically-patterned porous membranes with several user-defined pattern types. To demonstrate the utility of the patterned membranes, we selected one type of pattern and applied it to a membrane to serve as a cell culture support in a microfluidic model of a renal reabsorptive barrier. The topographic cues in the model resemble physiological cues found in vivo while the porous structure allows quantification of transport across the cell layer. Sub-micron surface topography generated via hot-embossing onto a track-etched polycarbonate membrane, fully replicated topographical features and preserved porous architecture. Pore size and shape were analyzed with SEM and image analysis to determine the effect of hot embossing on pore morphology. The membrane was assembled into a bilayer microfluidic device and a human kidney proximal tubule epithelial cell line (HK-2) and primary renal proximal tubule epithelial cells (RPTEC) were cultured to confluency on the membrane. Immunofluorescent staining of both cell types revealed protein expression indicative of the formation of a reabsorptive barrier responsive to mechanical stimulation: ZO-1 (tight junction), paxillin (focal adhesions) and acetylated α-tubulin (primary cilia). HK-2 and RPTEC aligned in the direction of ridge/groove topography of the membrane in the device, evidence that the device has mechanical control over cell response. This topographically-patterned porous membrane provides an in vitro platform on which to model reabsorptive barriers with meaningful applications for understanding biological transport phenomenon, underlying disease mechanisms, and drug toxicity.
Microphysiological organ-on-chip models offer the potential to improve the prediction of drug safety and efficacy through recapitulation of human physiological responses. The importance of including multiple cell types within tissue models has been well documented. However, the study of cell interactions in vitro can be limited by complexity of the tissue model and throughput of current culture systems. Here, we describe the development of a co-culture microvascular model and relevant assays in a high-throughput thermoplastic organ-on-chip platform, PREDICT96. The system consists of 96 arrayed bilayer microfluidic devices containing retinal microvascular endothelial cells and pericytes cultured on opposing sides of a microporous membrane. Compatibility of the PREDICT96 platform with a variety of quantifiable and scalable assays, including macromolecular permeability, image-based screening, Luminex, and qPCR, is demonstrated. In addition, the bilayer design of the devices allows for channel- or cell type-specific readouts, such as cytokine profiles and gene expression. The microvascular model was responsive to perturbations including barrier disruption, inflammatory stimulation, and fluid shear stress, and our results corroborated the improved robustness of co-culture over endothelial mono-cultures. We anticipate the PREDICT96 platform and adapted assays will be suitable for other complex tissues, including applications to disease models and drug discovery.
