design-development-three-dimensionally-printed-microscope-mask

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 ...

Texas Tech University

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.

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

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.

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

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.

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

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  norm of  was defined to characterize the difference between the cell trajectories and the associated streamlines. It was shown that  increases with micropost sizes and cell concentrations.

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

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.

Article	Year
A benchtop capillary flow layer-by-layer (CF-LbL) platform for rapid assembly and screening of biodegradable nanolayered films. Lab on a chip\| PubMed ID: 27785506	2016
Cell Isolation and Recovery Using Hollow Glass Microspheres Coated with Nanolayered Films for Applications in Resource-Limited Settings. ACS applied materials & interfaces\| PubMed ID: 28414907	2017
Enhanced capture and release of circulating tumor cells using hollow glass microspheres with a nanostructured surface. Nanoscale\| PubMed ID: 30160287	2018
Simulation of circulating tumor cell transport and adhesion in cell suspensions in microfluidic devices. Biomicrofluidics\| PubMed ID: 31737154	2019
Pulmonary-arterial-hypertension (PAH)-on-a-chip: fabrication, validation and application. Lab on a chip\| PubMed ID: 32749432	2020

Zhenya Ding

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