Effect of Surface Patterning and Presence of Collagen I on the Phenotypic Changes of Embryonic Stem Cell Derived Cardiomyocytes

Embryonic stem cell derived cardiomyocytes have been widely investigated for stem cell therapy or in vitro model systems. This study examines how two specific biophysical stimuli, collagen I and cell alignment, affect the phenotypes of embryonic stem cell derived cardiomyocytes in vitro. Three phenotypic indicators are assessed: sarcomere organization, cell elongation, and percentage of binucleation. Murine embryonic stem cells were differentiated in a hanging drop assay and cardiomyocytes expressing GFP-α-actinin were isolated by fluorescent sorting. First, the effect of collagen I was investigated. Addition of soluble collagen I markedly reduced binucleation as a result of an increase in cytokinesis. Laden with a collagen gel layer, myocyte mobility and cell shape change were impeded. Second, the effect of cell alignment by microcontact printing and nanopattern topography was investigated. Both patterning techniques induced cell alignment and elongation. Microcontact printing of 20 μm line pattern accelerated binucleation and nanotopography with 700 nm ridges and 3.5 μm grooves negatively regulated binucleation. This study highlights the importance of biophysical cues in the morphological changes of differentiated cardiomyocytes and may have important implications on how these cells incorporate into the native myocardium.Singapore-MIT Alliance for Research and TechnologyNational Science Foundation (U.S.) ((Science and Technology Center (EBICS): Emergent Behaviors of Integrated Cellular Systems, Grant CBET-0939511)Charles Stark Draper Laboratory (Internal Research and Development Program

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

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

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 A[subscript 3] 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.National Cancer Institute (U.S.) (Grant R33 CA174550-01)National Cancer Institute (U.S.) (Grant R21 CA140096)Italian Ministry of HealthCharles Stark Draper Laboratory (Fellowship

Hot embossing for fabrication of a microfluidic 3D cell culture

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.National Cancer Institute (U.S.) (award R21CA140096)Charles Stark Draper Laboratory (IR&D Grant

A microfluidic renal proximal tubule with active reabsorptive function

In the kidney, the renal proximal tubule (PT) reabsorbs solutes into the peritubular capillaries through active transport. Here, we replicate this reabsorptive function in vitro by engineering a microfluidic PT. The microfluidic PT architecture comprises a porous membrane with user-defined submicron surface topography separating two microchannels representing a PT filtrate lumen and a peritubular capillary lumen. Human PT epithelial cells and microvascular endothelial cells in respective microchannels created a PT-like reabsorptive barrier. Co-culturing epithelial and endothelial cells in the microfluidic architecture enhanced viability, metabolic activity, and compactness of the epithelial layer. The resulting tissue expressed tight junctions, kidney-specific morphology, and polarized expression of kidney markers. The microfluidic PT actively performed sodium-coupled glucose transport, which could be modulated by administration of a sodium-transport inhibiting drug. The microfluidic PT reproduces human physiology at the cellular and tissue levels, and measurable tissue function which can quantify kidney pharmaceutical efficacy and toxicity

Selection of Reserves for Woodland Caribou Using an Optimization Approach

Habitat protection has been identified as an important strategy for the conservation of woodland caribou (Rangifer tarandus). However, because of the economic opportunity costs associated with protection it is unlikely that all caribou ranges can be protected in their entirety. We used an optimization approach to identify reserve designs for caribou in Alberta, Canada, across a range of potential protection targets. Our designs minimized costs as well as three demographic risk factors: current industrial footprint, presence of white-tailed deer (Odocoileus virginianus), and climate change. We found that, using optimization, 60% of current caribou range can be protected (including 17% in existing parks) while maintaining access to over 98% of the value of resources on public lands. The trade-off between minimizing cost and minimizing demographic risk factors was minimal because the spatial distributions of cost and risk were similar. The prospects for protection are much reduced if protection is directed towards the herds that are most at risk of near-term extirpation

Polymer cell culture substrates with combined nanotopographical patterns and micropatterned chemical domains

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

An in vitro 3D microfluidic model mimics the reabsorptive barrier of the proximal tubule.

<p>A) In vivo, water and solutes cross an epithelial-endothelial barrier in the reabsorption process from filtrate tubule to the peritubular capillaries. (B) The microfluidic channels overlap to create a filtrate channel (green) in communication with a vascular channel (purple). The cross-sectional architecture (inset) mimics in vivo epithelial-endothelial barrier and generates cell-mediated transport through the membrane. (C) A cross-sectional SEM of the device shows a semi-porous membrane, which serves as a scaffold for the epithelial and endothelial cells and separates the filtrate and vascular channels. (D) The membrane sub-micron ridge/groove topography influences tissue organization and function.</p

The microfluidic PT model altered sodium-dependent reabsorption of glucose analog in response to ouabain.

<p>(A) hRPTEC expressed polarized transport proteins Na⁺/K⁺ ATPase and SGLT2 under co-culture conditions. (B) A schematic representation of experimental conditions. Under all conditions, the transport of a fluorescent glucose analog, 2-NBDG, from the filtrate channel (top) into the vascular channel (bottom) was observed using confocal z-stack and time-lapse microscopy to quantify intensity in the vascular channel. (C) 2-NBDG intensity versus time for one device subjected to all conditions sequentially shows the reduction in active transport due to ouabain administration. (D) The bar graph indicates the mean 2-NBDG intensity in the vascular channel at each condition, measured over ~15 minutes for 3 devices. Data are from 3 replicates of the experiment. Error bars represent standard error of the main effects as computed from the error term in the analysis of covariance model. Introducing ouabain to the vascular channel blocked 2-NBDG transport. The 2-NBDG transport recovered when ouabain was rinsed from the system. A second administration of ouabain again blocked 2-NBDG transport. The tissue recovery and repeat effect of ouabain demonstrates dynamic reabsorptive cell-mediated reabsorption function. * P < 0.0005. Experimental results were verified for 3 independent experiments.</p