Microfabrication for Biomedical Research

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The use of microfabricated and microfluidic structures in biomedical research continues to expand, but many research applications for these devices require customization, and new applications typically require several design iterations for troubleshooting and optimization. In an effort to bring the necessary technical capabilities and knowledge to do so into biomedical laboratories, we have developed a basic in-house microfabrication facility accessible to researchers across the intramural program. Although the resolution, device yield, and complexity are somewhat lower than those achievable with a dedicated cleanroom, they are nonetheless sufficient for many experiments on cells. Furthermore, the instrumentation complexity, fabrication cost, and turnaround time are greatly reduced, which enables rapid cycling through design parameters and decreases the time needed to train new users. We are able to reliably pattern single- and multi-layer template features with lateral dimensions of less than 2 microns, and with heights ranging from a few microns to a few hundred microns. We have developed protocols for using these templates to generate microstructured PDMS, agarose, and PEGDA hydrogels, including techniques for making and manipulating thin (<200 micrometer) PDMS layers for use in multilayer devices and bottomless structures. We can also perform surface modification of PDMS and other polymers, including the irreversible bonding of PDMS to glass, and have developed techniques for connecting devices to flow-control instruments such as syringe pumps and pressure controllers. We have protocols for generating micropatterns of biomolecules on surfaces using microcontact printing or selective UV exposure, as well as protocols for generating monodisperse droplets and hydrogel beads. In addition, we have the ability to deposit and pattern metal layers, as well as a heated hydraulic press for hot-embossing thermoplastics, including PMMA, polycarbonate, and COC. We also have a programmable razor cutter which we can use with pressure-sensitive adhesive to directly make thin film structures with heights ranging from 25 to a few hundred microns, and sub-millimeter lateral dimensions. This is a low-cost and convenient method for several applications, including the ready fabrication of flow cells with two glass walls, or for fluidic confinement over already-functionalized surfaces. We recently installed a simple extrusion bioprinter, which adds the capability to print hydrogels and sacrificial materials that can then be encased in hydrogels; the system is installed in a biosafety cabinet to enable printing of cell-laden materials as needed. As a companion tool as well as to support expanding interest in biomaterials development within the IRP, we have also acquired a rotational rheometer capable of characterizing liquids and gels over a temperature range of 1-60 degrees Celsius. Finally, we continue to work on finite element modeling of transport in microfabricated structures, developing models for oxygen delivery in a bioreactor with micropillars and for chemokine concentration in a microfluidic hydrogel device. These capabilities have found application in a number of projects, representing a broad variety of interests and institutes. In addition to the representative projects discussed below and others still in the early stages, we have also trained researchers in basic microfabrication techniques, including personnel from other laboratories in NEI, NIAID, NIAMS, NHLBI, NCI, NICHD, NINDS, NIDCD, NIDDK, NCATS, and NIBIB. Some examples of recent and ongoing projects are: 1) A project with the BRS, VRC, NIAID to improve the throughput and reliability of their droplet-based yeast display system for interrogating antibody libraries in order to probe humoral immune responses to vaccination and natural infection. Past work with the technology has looked at responses to Ebola vaccination, and the system is intended to look at patient responses to Sars-cov2 vaccines. Our initial work has focused on fabricating a simple microfluidic flow-focusing droplet generator for coencapsulation of B-cells and superparamagnetic oligo beads; current efforts are extending this design to parallel nozzles for faster processing of single samples and parallel processing of multiple samples. 2) The design, fabrication, modeling, and use of an oxygen-transmissive membrane, patterned with a micropillar array to deliver oxygen to three-dimensional cell culture volumes with in vivo-like spatial distribution, in collaboration with LCB, CCR, NCI and SPIS, CIT. Because the pillar spacing is approximately equal to typical intercapillary distances (200 microns), cells in a Matrigel layer surrounding the pillars can be maintained under hypoxic conditions in an extended 3D volume. We are also continuing to work with researchers in LAMB, NHLBI to employ their recently developed FLIM-FRET probe for intracellular oxygen to directly image spatial and cellular variations in oxygenation in these structures, and have started applying finite element modeling to understand the interaction between transport and consumption at these length scales. 3) A collaborative project with researchers in LBC, NIDDK, to implement their existing PNA-based assay on a microfluidic platform and using gold nanoparticles to provide an optical readout with minimal instrumentation. 4) The use of microstructured PDMS to pattern surfaces, such as an application for controlling cellular attachment with researchers in LGI, NCI who are using Matrigel confinement of cellular clusters to promote differentiation in stem cells. 5) A collaborative project with SI, NIBIB to develop a microfluidic device for studying the foreign body response in co-cultures of fibroblasts and macrophages. 6) A collaborative effort with LSB, NIAID, to study chemotaxis, currently focusing on the decision-making of single cells exposed to competing solution gradients. Earlier efforts with this group looked at chemotaxis of primary immune cells in 3-D collagen matrices, using a microfluidic agarose device to generate reproducible time-varying spatial gradients on a platform compatible with high-resolution fluorescence and two-photon imaging. 7) In collaboration with LCB, NCI, development of in vitro models to complement measurements on cell migration in zebrafish to advance understanding of the mechanisms for cell migration and metastasis. 8) Ongoing efforts with researchers from the MBS, NCI to assist in their development of top-down in vitro models of metastasis using tissues resected from patients undergoing surgery. 9) In collaboration with researchers in NINDS and NICHD, the development of devices to enable selective harvesting of axons from cultured neurons for biochemical analysis, in order to readily be able to generate enough material for downstream measurements. These large-volume, high-density devices have found use in a number of laboratories across the IRP. 10) A collaboration with LCTM, NHLBI to fabricate artificial vessels for studying the effects of vessel geometry, particularly curvature, on cell mechanics. An extrusion bioprinter is used to print sacrificial structures in Pluronic F-127 on a hydrogel base, after which the structures are immediately encased in hydrogel, chilled, and washed away before introducing cells to the open vessel that remains. 11) A project with HROI, NIBIB to investigate the use of a newly available silicone elastomer (BIO-133), with a refractive index similar to that of water, in fabricating microstructured devices compatible with superresolution imaging. 12) With researchers in NICHD, development of a device for co-culture of granulosa and theca cells in alginate-shell collagen-core capsules, aimed at developing an in vitro ovarian follicle.