Engineering and Characterization of a Biomimetic Microchip for Differentiating Mouse Adipocytes in a 3D Microenvironment.

Although two-dimensional (2D) cell cultures are the standard in cell research, one pivotal disadvantage is the lack of cell-cell and cell-extracellular matrix (ECM) signaling in the culture milieu. However, such signals occur in three-dimensional (3D) in vivo environments and are essential for cell differentiation, proliferation, and a range of cellular functions. In this study, we developed a microfluidic device to proliferate and differentiate functional adipose tissue and adipocytes by utilizing 3D cell culture technology. This device was used to generate a tissue-specific 3D microenvironment to differentiate 3T3-L1 preadipocytes into either visceral white adipocytes using visceral adipose tissue (VAT) or subcutaneous white adipose tissue (SAT). The microchip has been tested and validated by functional assessments including cell morphology, inflammatory response to a lipopolysaccharide (LPS) challenge, GLUT4 tracking, and gene expression analyses. The biomimetic microfluidic chip is expected to mimic functional adipose tissues that can replace 2D cell cultures and allow for more accurate analysis of adipose tissue physiology.

Design and Development of a Three-Dimensionally Printed Microscope Mask Alignment Adapter for the Fabrication of Multilayer Microfluidic Devices.

This project aims to develop an easy-to-use and cost-effective platform for the fabrication of precise, multilayer microfluidic devices, which typically can only be achieved using costly equipment in a clean room setting. The key part of the platform is a three dimensionally (3D) printed microscope mask alignment adapter (MMAA) compatible with regular optical microscopes and ultraviolet (UV) light exposure systems. The overall process of creating the device has been vastly simplified because of the work done to optimize the device design. The process entails finding the proper dimensions for the equipment available in the laboratory and 3D-printing the MMAA with the optimized specifications. Experimental results show that the optimized MMAA designed and manufactured by 3D printing performs well with a common microscope and light exposure system. Using a master mold prepared by the 3D-printed MMAA, the resulting microfluidic devices with multilayered structures contain alignment errors of <10 µm, which is sufficient for common microchips. Although human error through transportation of the device to the UV light exposure system can cause larger fabrication errors, the minimal errors achieved in this study are attainable with practice and care. Furthermore, the MMAA can be customized to fit any microscope and UV exposure system by making changes to the modeling file in the 3D printing system. This project provides smaller laboratories with a useful research tool as it only requires the use of equipment that is typically already available to laboratories that produce and use microfluidic devices. The following detailed protocol outlines the design and 3D printing process for the MMAA. In addition, the steps for procuring a multilayer master mold using the MMAA and producing poly(dimethylsiloxane) (PDMS) microfluidic chips is also described herein.

Direct monitoring of protease activity using an integrated microchip coated with multilayered fluorogenic nanofilms.

Proteases play an essential role in the four sequential but overlapping phases of wound healing: hemostasis, inflammation, proliferation, and remodeling. In chronic wounds, excessive protease secretion damages the newly formed extracellular matrix, thereby delaying or preventing the normal healing process. Peptide-based fluorogenic sensors provide a visual platform to sense and analyze protease activity through changes in the fluorescence intensity. Here, we have developed an integrated microfluidic chip coated with multilayered fluorogenic nanofilms that can directly monitor protease activity. Fluorogenic protease sensors were chemically conjugated to polymer films coated on the surface of parallel microfluidic channels. Capillary flow layer-by-layer (CF-LbL) was used for film assembly and combined with subsequent sensor modification to establish a novel platform sensing technology. The benefits of our platform include facile fabrication and processing, controllable film nanostructure, small sample volume, and high sensitivity. We observed increased fluorescence of the LbL nanofilms when they were exposed to model recombinant proteases, confirming their responsiveness to protease activity. Increases in the nanofilms' fluorescence intensity were also observed during incubation with liquid extracted from murine infected wounds, demonstrating the potential of these films to provide real-time, in situ information about protease activity levels.

Pulmonary-arterial-hypertension (PAH)-on-a-chip: fabrication, validation and application.

Currently used animal and cellular models for pulmonary arterial hypertension (PAH) only partially recapitulate its pathophysiology in humans and are thus inadequate in reproducing the hallmarks of the disease, inconsistent in portraying the sex-disparity, and unyielding to combinatorial study designs. Here we sought to deploy the ingenuity of microengineering in developing and validating a tissue chip model for human PAH. We designed and fabricated a microfluidic device to emulate the luminal, intimal, medial, adventitial, and perivascular layers of a pulmonary artery. By growing three types of pulmonary arterial cells (PACs)-endothelial, smooth muscle, and adventitial cells, we recreated the PAH pathophysiology on the device. Diseased (PAH) PACs, when grown on the chips, moved of out their designated layers and created phenomena similar to the major pathologies of human PAH: intimal thickening, muscularization, and arterial remodeling and show an endothelial to mesenchymal transition. Flow-induced stress caused control cells, grown on the chips, to undergo morphological changes and elicit arterial remodeling. Our data also suggest that the newly developed chips can be used to elucidate the sex disparity in PAH and to study the therapeutic efficacy of existing and investigational anti-PAH drugs. We believe this miniaturized device can be deployed for testing various prevailing and new hypotheses regarding the pathobiology and drug therapy in human PAH.

Simulation of circulating tumor cell transport and adhesion in cell suspensions in microfluidic devices.

Understanding cell transport and adhesion dynamics under flow is important for many biotransport problems. We investigated the influence of cell size, ligand coating density, micropost size, and intercellular collisions on circulating tumor cell adhesion and transport in microfluidic devices. The cells were modeled as coarse-grained cell membranes and the adhesion was modeled as pairwise interacting potentials, while the fluid was solved using the lattice Boltzmann method. The coupling between the cell and the fluid was achieved through the immersed boundary method. The cell showed transient rolling adhesion in high shear regions and firm adhesion in low shear regions. The adhesive force for rolling cells on a micropost was increasing before the cell reached the crest of the post and then decreasing afterward. The adhesive strength for cells increases with ligand coating density. Cell trajectories in a microfluidic device with a shifted post design were studied as well. At low concentrations, the majority of the cells follow streamlines closely. However, the intercellular collision and collision from red blood cells impacted the cell trajectories. An L 2 norm of | e | was defined to characterize the difference between the cell trajectories and the associated streamlines. It was shown that | e | L 2 increases with micropost sizes and cell concentrations.

Microfluidic preparation, shrinkage, and surface modification of monodispersed alginate microbeads for 3D cell culture.

Functionalized alginate microbeads (MB) have been widely used for three-dimensional (3D) culture of cells and creating biomimetic tissue models. However, conventional methods for preparing these MB suffer from poor polydispersity, due to coalescence of droplets during the gelation process and post-aggregation. It remains an immense challenge to prepare alginate MB with narrow size distribution and uniform shape, especially when their diameters are similar to the size of cells. In this work, we developed a simple method to produce monodispersed, cell-size alginate MB through microfluidic emulsification, followed by a controlled shrinkage process and gelation in mineral oil with low concentration of calcium ion (Ca2+). During the gelation process caused by the diffusion of Ca2+ from the oil to water phase, a large amount of satellite droplets with sub-micrometer sizes was formed at the water/oil interface. As a result, each original droplet was transformed to one shrunken-MB with much smaller size and numerous submicron-size satellites. To explore the feasibility of the shrunken-MB for culturing with cells, we have successfully modified a variety of polymer nanofilms on MB surfaces using a layer-by-layer assembly approach. Finally, the nanofilm-modified MB was applied to a 3D culture of GFP-expressing fibroblast cells and demonstrated good biocompatibility.

Effective reduction of non-specific binding of blood cells in a microfluidic chip for isolation of rare cancer cells.

The high purity of target cells enriched from blood samples plays an important role in the clinical detection of diseases. However, non-specific binding of blood cells in the isolated cell samples can complicate downstream molecular and genetic analysis. In this work, we report a simple solution to non-specific binding of blood cells by modifying the surface of microchips with a multilayer nanofilm, with the outmost layer containing both PEG brushes for reducing blood cell adhesion and antibodies for enriching target cells. This layer-by-layer (LbL) polysaccharide nanofilm was modified with neutravindin and then conjugated with a mixture of biotinylated PEG molecules and biotinylated antibodies. Using EpCAM-expressing and HER2-expressing cancer cells in blood as model platforms, we were able to dramatically reduce the non-specific binding of blood cells to approximately 1 cell per mm2 without sacrificing the high capture efficiency of the microchip. To support the rational extension of this approach to other applications for cell isolation and blood cell resistance, we conducted extensive characterization on the nanofilm formation and degradation, antifouling with PEG brushes and introducing functional antibodies. This simple, yet effective, approach can be applied to a variety of microchip applications that require high purity of sample cells containing minimal contamination from blood cells.

Enhanced capture and release of circulating tumor cells using hollow glass microspheres with a nanostructured surface.

Self-floating hollow glass microspheres (HGMS) modified with tumor-specific antibodies have been developed for the capture of circulating tumor cells (CTCs), and have demonstrated effective cell isolation and good viability of isolated cancer cells. However, the capture efficiency decreases dramatically if the spiked cell concentration is low, possibly due to insufficient interactions between cancer cells and the HGMS surface. In order to apply HGMS-based CTC isolation to clinically relevant samples, it is desirable to create nanostructures on the surface of HGMS to enhance cell-surface interactions. Nevertheless, current microfabrication methods cannot generate nanostructured-surfaces on microspheres. The authors have developed a new HGMS with a controlled nanotopographical surface structure (NSHGMS), and demonstrated isolation and recovery of rare cancer cells. NSHGMS are achieved by applying layer-by-layer (LbL) assembly of negatively charged SiO2 nanoparticles and positively charged poly-l-arginine molecules, then sheathing the surface with an enzymatically degradable LbL film made from biotinylated alginate and poly-l-arginine, and capping with anti-EpCAM antibodies and anti-fouling PEG molecules. Compared to smooth-surfaced HGMS, NSHGMS showed shorter isolation time (20 min), enhanced capture efficiency (93.6 ± 4.9%) and lower detection limit (30 cells per mL) for commonly used cancer cell lines (MCF7, SK-BR-3, PC-3, A549 and CCRF-CEM). This NSHGMS-based CTC isolation method does not require specialized lab equipment or an external power source, and thus, can be used for the separation of targeted cells from blood or other body fluids in a resource-limited environment.

Cell Isolation and Recovery Using Hollow Glass Microspheres Coated with Nanolayered Films for Applications in Resource-Limited Settings.

Established cell isolation and purification techniques such as fluorescence-activated cell sorting (FACS), isolation through magnetic micro/nanoparticles, and recovery via microfluidic devices have limited application as disposable technologies appropriate for point-of-care use in remote areas where lab equipment as well as electrical, magnetic, and optical sources are restricted. We report a simple yet effective method for cell isolation and recovery that requires neither specialized lab equipment nor any form of power source. Specifically, self-floating hollow glass microspheres were coated with an enzymatically degradable nanolayered film and conjugated with antibodies to allow both fast capture and release of subpopulations of cells from a cell mixture. Targeted cells were captured by the microspheres and allowed to float to the top of the hosting liquid, thereby isolating targeted cells. To minimize nonspecific adhesion of untargeted cells and to enhance the purity of the isolated cell population, an antifouling polymer brush layer was grafted onto the nanolayered film. Using the EpCAM-expressing cancer cell line PC-3 in blood as a model system, we have demonstrated the isolation and recovery of cancer cells without compromising cell viability or proliferative potential. The whole process takes less than 1 h. To support the rational extension of this platform technology, we introduce extensive characterization of the critical design parameters: film formation and degradation, grafting with a poly(ethylene glycol) (PEG) sheath, and introducing functional antibodies. Our approach is expected to overcome practical hurdles and provide viable targeted cells for downstream analyses in resource-limited settings.

A benchtop capillary flow layer-by-layer (CF-LbL) platform for rapid assembly and screening of biodegradable nanolayered films.

Capillary flow layer-by-layer (CF-LbL) is a microfluidic platform for high throughput preparation and screening of nanolayered polymer films. Using a simple benchtop version of CF-LbL, we systematically studied the effects of various flow conditions and channel geometries on the thickness and surface roughness of the resulting films. We also investigated the biocompatibility and degradation behaviors of a series of enzymatically-degradable films made from naturally derived polymers, i.e. either alginate or hyaluronic acid as the anionic species and poly-l-arginine as the positive species. Furthermore, using one optimized film formulation for coating on the inside walls of a microfluidic chip, we successfully demonstrated the ability of this film to capture and rapidly release cancer cells from whole blood. This simple platform is expected to be a powerful tool to increase the accessibility of the LbL film assembly to a broader scientific community.

4-(Cyclo-propane-carboxamido)-benzoic acid.

In the title compound, C(11)H(11)NO(3), the dihedral angle between the benzene ring and the cyclo-propane ring is 63.2 (1)°. In the crystal, mol-ecules are linked through classical cyclic carb-oxy-lic acid O-H⋯O hydrogen-bond inter-actions [graph set R(2) (2)(8)] giving centrosymmetric dimers which are extended along the b-axis direction through amide N-H⋯O hydrogen-bond inter-actions, giving one-dimensional ribbon structures. Weak C-H⋯O inter-actions are also present in the structure.