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Principal Investigator: Nicole Y Morgan
Organization: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING
Fiscal Year: 2024
Award: $1,510,844
Funding agency: National Institute of Biomedical Imaging and Bioengineering
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.
In addition, we have 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 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, NIDDK, NCATS, and NIBIB. Some examples of recent and ongoing projects are:
1) A project with VPDS, IML, NIAID to implement FIND-Seq, a technique for performing transcriptomics on rare cells that cannot be identified with surface markers. 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; to facilitate their adoption we have made epoxy molds which combine millimeter-scale reservoirs and micrometer-scale 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 have developed a 24-well plate format as well as a single chamber device that is compatible with extended real-time imaging. 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. 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) Work with ABU, LI, NIAID to employ a microfluidic device to encapsulate cells in double emulsions compatible with FACS analysis for subsequent study of rare cells, using fluorescence localization on co-encapsulated beads to screen for specific secreted antibodies.
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) 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) A collaborative project with SI, NIBIB to develop a microfluidic device for studying the foreign body response in co-cultures of fibroblasts and macrophages.
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 exosome capture devices for image-based analysis of 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 the newly established MATRICES lab, NIBIB, to develop in vitro models of carotid bifurcations for studying ECM remodeling in sickle cell disease.
Terms: <3-D><3-Dimensional><3D><3D cell culture><3D culture><Adoption><Agarose><Antibodies><Award><Axon><Back><Biochemical><Biomaterials Research><Biomedical Research><Body Tissues><Brachydanio rerio><CCR><COVID-19 infection><COVID-19 virus infection><COVID19 infection><Cell Body><Cell Locomotion><Cell Migration><Cell Movement><Cell-Extracellular Matrix><Cells><Cellular Migration><Cellular Motility><Chemotactic Cytokines><Chills><Clinical><Co-culture><Cocultivation><Coculture><Coculture Techniques><Collaborations><Companions><Complement><Complement Proteins><Consumption><Danio rerio><Dedications><Development><Device Designs><Device or Instrument Development><Devices><Dimensions><Discipline><Dorsum><ECM><Elements><Emulsions><Encapsulated><Endothelium><Equipment><Exposure to ultraviolet radiation><Extracellular Matrix><FRET><Fibroblasts><Filamentous Fungi><Fluorescence><Fluorescence Resonance Energy Transfer><Foreign Bodies><Förster Resonance Energy Transfer><Gel><Geometry><Glass><Harvest><Hb SS disease><HbSS disease><Height><Hemoglobin S Disease><Hemoglobin sickle cell disease><Hemoglobin sickle cell disorder><High Throughput Assay><Homologous Chemotactic Cytokines><Human Resources><Hydrogels><Hypoxia><Hypoxic><Image><Imaging Device><Imaging Instrument><Imaging Tool><Intercrines><Intramural Program><Intramural Research Program><Investigators><Knowledge><Laboratories><Leanness><Length><Liquid substance><Macrophage><Manpower><Measurement><Mechanics><Membrane><Metastasis><Metastasize><Metastatic Lesion><Metastatic Mass><Metastatic Neoplasm><Metastatic Tumor><Microfabrication><Microfluidic Device><Microfluidic Lab-On-A-Chip><Microfluidic Microchips><Microfluidics><Modeling><Modification><Molds><Mφ><NCATS><NHLBI><NIAID><NIAMS><NIBIB><NICHD><NIDCD><NIDDK><NIH><NINDS><National Center for Advancing Translational Sciences><National Heart, Lung, and Blood Institute><National Institute of Allergy and Infectious Disease><National Institute of Arthritis, and Musculoskeletal, and Skin Diseases><National Institute of Biomedical Imaging and Bioengineering><National Institute of Child Health and Human Development><National Institute of Children's Health and Human Development><National Institute of Diabetes and Digestive and Kidney Diseases><National Institute of Neurological Diseases and Stroke><National Institute of Neurological Disorders and Stroke><National Institute on Deafness and Other Communication Disorders><National Institutes of Health><Neoplasm Metastasis><Nerve Cells><Nerve Unit><Neural Cell><Neurocyte><Neurons><O element><O2 element><Operative Procedures><Operative Surgical Procedures><Oxygen><Oxygen Deficiency><PEG-DA><Patients><Pattern><Pluronics><Polymers><Printing><Process><Progenitor Cells><Protocol><Protocols documentation><Pump><Research><Research Personnel><Researchers><Resected><Resolution><Resource Sharing><Robotics><SARS-CoV-2 infection><SARS-CoV2 infection><SIS cytokines><Sampling><Secondary Neoplasm><Secondary Tumor><Sepharose><Severe acute respiratory syndrome coronavirus 2 infection><Sickle Cell Anemia><Spatial Distribution><Structure><Surface><Surgical><Surgical Interventions><Surgical Procedure><Syringes><System><Techniques><Temperature><Thinness><Time><Tissues><Training><UV Radiation Exposure><UV exposure><Ultraviolet Radiation Related Exposure><Ultraviolet radiation exposure><United States National Institutes of Health><With laterality><Work><Zebra Danio><Zebra Fish><Zebrafish><base><bases><bench bed side><bench bedside><bench to bed side><bench to bedside><bench to clinic><bench to clinical practice><biological systems><biomaterial development><cancer metastasis><cell motility><chemoattractant cytokine><chemokine><complementation><coronavirus disease 2019 infection><density><design><designing><developmental><device development><exosome><experiment><experimental research><experimental study><experiments><fabrication><fabrication cost><fluid><high throughput screening><imaging><in vitro Model><in vivo><infected with COVID-19><infected with COVID19><infected with SARS-CoV-2><infected with SARS-CoV2><infected with coronavirus disease 2019><infected with severe acute respiratory syndrome coronavirus 2><instrument><instrument development><instrumentation><interest><liposomal formulation><liposomal preparation><liquid><manufacturing cost><matrigel><mechanic><mechanical><membrane structure><meter><microfluidic chip><millimeter><neuronal><operation><operations><personnel><poly(ethylene glycol)diacrylate><poly(ethyleneglycol) diacrylate><polyethyleneglycol diacrylate><polymer><polymeric><pressure><prototype><pulmonary arterial hypertension><pulmonary artery hypertension><real-time images><realtime image><resolutions><response><senescence><senescent><sickle cell disease><sickle cell disorder><sickle disease><sicklemia><simulation><stem cells><surgery><three dimensional><three dimensional cell culture><tissue culture><tool><transcriptomics><tumor cell metastasis><ultraviolet exposure><ultraviolet light exposure><viscoelasticity><µfluidic>