Microfabrication for Biomedical Research

National Institute Of Biomedical Imaging And Bioengineering, Bethesda

Investigators

Linked publications, trials & patents

Abstract

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. To help bring the necessary technical capabilities and knowledge 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. For projects that require more sophisticated capabilities, we can help facilitate access to and use of multiuser fabrication facilities nearby. 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 established 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. We have also acquired a simple extrusion bioprinter to add 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 and consumption in tissue culture 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, NIDCR, NIDDK, NCATS, NHGRI, CC, and NIBIB. Some examples of recent and ongoing projects are: 1) A project with VPDS, IL, VRC, NIAID to implement FIND-Seq, a technique for performing transcriptomics on rare cells that cannot be identified with surface markers. Instrumentation for two of the three steps has been installed in the VPDS laboratory already; the third is under development within MMU. This project received an NIH Directors Challenge award in 2022. 2) 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, including three groups in NICHD, one in NINDS, and one in NHGRI; to facilitate their adoption we have made epoxy molds which combine millimeter-scale reservoirs and micrometer-scale axonal confinement channels. 3) 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. This year we continued to refine the single-use 24-well plate format and develop protocols for extended real-time imaging and cell-tracking in the single chamber device format. We continue to work with researchers in LAMB, NHLBI to employ their FLIM-FRET probes to directly image intracellular oxygenation in these devices, and have used finite element modeling to understand the interaction between transport and consumption at these length scales. 4) A collaborative project with researchers in CCMD, CC to adapt their in vitro model for pulmonary arterial hypertension to study endothelial senescence with SARS-Cov-2 infection in a multicellular tissue chip device. This project received a bench to bedside and back award. 5) 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. 6) A new project with CBBS, NINDS, to micropattern substrates using detachable microchannels for use in cell motility studies. 7) In collaboration with LCB, NCI, development of in vitro models to complement their in vivo measurements in a zebrafish model in order to advance understanding of the mechanisms for cell migration and metastasis. As part of this work, we have also assisted with bulk viscoelasticity measurements on extracellular matrix mimics. 8) A project with SBS, NIDCR to develop protocols for controlled fabrication of functionalized fibrin microbeads to facilitate cartilage regeneration 9) A collaborative project with UNEMPS, NIBIB to develop a microfluidic thyroid model. 10) A project with VPPL, NIAID to develop microfluidic devices that can be interfaced directly with their robotic pipetting stations for moderately high-throughput screening of liposome formulations. 11) A project with NINDS/CARD to implement a recirculation capture step in surface-functionalized microfluidic devices for image-based analysis of exosomes in clinical CSF samples. 12) 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. 13) A project with RCBD, NNRL, NEI to assist with computational and experimental characterization of flow in a novel bioreactor for retinal organoid culture. 14) A project with MATRICES, NIBIB, to implement a microfluidic device for separation and concentration of white blood cells for enzymatic analysis in a low resource setting.