26 research outputs found
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Defining cellular microenvironments using multiphoton lithography
textTo understand the chemistry of life processes in detail is largely a challenge of resolving them in their native, cellular environment. Cell culture, first developed a century ago, has proven to be an essential tool for reductionist studies of cellular biochemistry and development. However, for the technology of cell culture to move forward and address increasingly complex problems, in vitro environments must be refined to better reflect the cellular environment in vivo. This dissertation work has focused on the development of methods to define cellular microenvironments using the high resolution, 3D capabilities of multiphoton lithography. Here, site-specific photochemistry using multiphoton excitation is applied to the photocrosslinking of proteins, providing the means to organize bioactive species into well-defined 3D microenvironments. Further, conditions have been identified that enable microfabrication to be performed in the presence of cells -- allowing cell outgrowth and motility to be directed in real time. In addition to the intrinsic chemical functionality of microfabricated protein structures, 3D protein matrices are shown to respond mechanically to changes in the chemical environment, enabling new avenues for micro-scale actuation to be explored. Complex 2D and 3D protein photocrosslinking is further facilitated by integrating transparency and automated reflectance photomasks into the fabrication system. These advances could be transformative in efforts to fabricate precise cellular scaffolding that replicates the morphological (and potentially biochemical) features of in vivo tissue microenvironments. Finally, these methods are applied to the study of microorganism behavior with single-cell resolution. Microarchitectures are designed that allow the position and motion of motile bacterial to generate directional microfluidic flow -- providing a foundation to develop micro-scale devices powered by cells.Chemistry and BiochemistryBiochemistr
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Direct Writing and Actuation of Three-Dimensionally Patterned Hydrogel Pads on Micropillar Supports
Freely swelling, three-dimensionally patterned responsive hydrogels fabricated by multiphoton lithography on the tips of flexible pillars provide unique capabilities for the design of adaptive systems. The resulting materials have tunable actuation direction and angle, sensitive optical response, and precise spatial integration of gels with varying pH and temperature response (see picture; scale bar: 20 μm).Engineering and Applied Science
Silica bioreplication preserves three-dimensional spheroid structures of human pluripotent stem cells and HepG2 cells
Three-dimensional (3D) cell cultures produce more in vivo-like multicellular structures such as spheroids that cannot be obtained in two-dimensional (2D) cell cultures. Thus, they are increasingly employed as models for cancer and drug research, as well as tissue engineering. It has proven challenging to stabilize spheroid architectures for detailed morphological examination. Here we overcome this issue using a silica bioreplication (SBR) process employed on spheroids formed from human pluripotent stem cells (hPSCs) and hepatocellular carcinoma HepG2 cells cultured in the nanofibrillar cellulose (NFC) hydrogel. The cells in the spheroids are more round and tightly interacting with each other than those in 2D cultures, and they develop microvilli-like structures on the cell membranes as seen in 2D cultures. Furthermore, SBR preserves extracellular matrix-like materials and cellular proteins. These findings provide the first evidence of intact hPSC spheroid architectures and similar fine structures to 2D-cultured cells, providing a pathway to enable our understanding of morphogenesis in 3D cultures.Peer reviewe
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Multiphoton Lithography of Nanocrystalline Platinum and Palladium for Site-Specific Catalysis in 3D Microenvironments
Integration of catalytic nanostructured platinum and palladium within 3D microscale structures or fluidic environments is important for systems ranging from micropumps to microfluidic chemical reactors and energy converters. We report a straightforward procedure to fabricate microscale patterns of nanocrystalline platinum and palladium using multiphoton lithography. These materials display excellent catalytic, electrical, and electrochemical properties, and we demonstrate high-resolution integration of catalysts within 3D defined microenvironments to generate directed autonomous particle and fluid
transport.Engineering and Applied Science
Development and characterization of 3D, nano-confined multicellular constructs for advanced biohybrid devices.
This is the final report for the President Harry S. Truman Fellowship in National Security Science and Engineering (LDRD project 130813) awarded to Dr. Bryan Kaehr from 2008-2011. Biological chemistries, cells, and integrated systems (e.g., organisms, ecologies, etc.) offer important lessons for the design of synthetic strategies and materials. The desire to both understand and ultimately improve upon biological processes has been a driving force for considerable scientific efforts worldwide. However, to impart the useful properties of biological systems into modern devices and materials requires new ideas and technologies. The research herein addresses aspects of these issues through the development of (1) a rapid-prototyping methodology to build 3D bio-interfaces and catalytic architectures, (2) a quantitative method to measure cell/material mechanical interactions in situ and at the microscale, and (3) a breakthrough approach to generate functional biocomposites from bacteria and cultured cells
Biocompatible Microfabrication of 3D Isolation Chambers for Targeted Confinement of Individual Cells and Their Progeny
We describe a technique to physically isolate single/individual
cells from their surrounding environment by fabricating three-dimensional
microchambers around selected cells under biocompatible conditions.
Isolation of targeted cells is achieved via rapid fabrication of protein
hydrogels from a biocompatible precursor solution using multiphoton
lithography, an intrinsically 3D laser direct write microfabrication
technique. Cells remain chemically accessible to environmental cues
enabling their propagation into well-defined, high density populations.
We demonstrate this methodology on gram negative (<i>E. coli</i>), gram positive (<i>S. aureus</i>), and eukaryotic (<i>S. cerevisiae</i>) cells. The opportunities to confine viable,
single/individual-cells and small populations within user-defined
microenvironments afforded by this approach should facilitate the
study of cell behaviors across multiple generations