Rice University View Institution's Website 21 articles published in JoVE Bioengineering Fabrication of Pulsatile Polymeric Microparticles Encapsulating Rabies Antigen Tyler P. Graf*1, Kadryn Kadasia*2, Sarah Melhorn1, Eliza Kessler3, Haisong Yang2, Tsvetelina Baryakova1, Samantha Brady2, Kevin J. McHugh1,4 1Department of Bioengineering, Rice University, 2Particles for Humanity, 3IMPACT Technology Development, 4Department of Chemistry, Rice University This method describes the encapsulation of the rabies antigen into biodegradable polymeric microparticles with structural and material properties that enable pulsatile release after a predetermined delay. Enzyme-linked immunosorbent assay (ELISA) assessment of the antigen retrieved from the particle core confirms the presence of intact trimeric rabies virus glycoprotein through particle fabrication. Developmental Biology Investigating Scarless Tissue Regeneration in Embryonic Wounded Chick Corneas Manish Pathuri1, James Spurlin III2, Peter Lwigale2, Tyler Schwend1 1Department of Biology, Illinois Wesleyan University, 2Department of Biosciences, Rice University The present protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. The regenerating or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure. Bioengineering Open-Source Miniature Fluorimeter to Monitor Real-Time Isothermal Nucleic Acid Amplification Reactions in Resource-Limited Settings Jackson Coole*1, Alex Kortum*1, Yubo Tang1, Imran Vohra1, Yajur Maker1, Kathryn Kundrod1, Mary Natoli1, Rebecca Richards-Kortum1 1Department of Bioengineering, Rice University Detailed instructions are provided to build an open-source, modular fluorimeter that is compatible with many low-cost heaters to perform real-time, quantitative isothermal nucleic acid amplification. Neuroscience Focused Ultrasound Induced Blood-Brain Barrier Opening for Targeting Brain Structures and Evaluating Chemogenetic Neuromodulation Jerzy O. Szablowski1, Manwal Harb1 1Department of Bioengineering, Rice University This protocol delineates steps necessary for the gene delivery through focused ultrasound blood brain barrier (BBB) opening, evaluation of the resulting gene expression, and measurement of neuromodulation activity of chemogenetic receptors through histological tests. Cancer Research Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform Lindsey K. Sablatura1, Kristin M. Bircsak2, Peter Shepherd3, Karla Queiroz4, Mary C. Farach-Carson1,5,6, Pamela E. Constantinou1,7, Anthony Saleh2, Nora Navone3, Daniel A. Harrington1,5,6 1Department of BioSciences, Rice University, 2Mimetas US Inc, 3Department of Genitourinary Medical Oncology Research, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, 4Mimetas B.V., 5Department of Bioengineering, Rice University, 6Department of Diagnostic and Biomedical Sciences, The University of Texas Health Science Center, 7Sheikh Ahmed Center for Pancreatic Cancer Research, The University of Texas MD Anderson Cancer Center This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture in a perfused microfluidic platform. Cancer Research An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity Jingqi Pei*1, Svetlana B. Panina*1, Natalia V. Kirienko1 1Department of BioSciences, Rice University The protocol describes a rapid, high-throughput, reliable, inexpensive, and unbiased assay for efficiently determining cellular viability. This assay is particularly useful when cells' mitochondria have been damaged, which interferes with other assays. The assay uses automated counting of cells stained with two nuclear dyes – Hoechst 33342 and propidium iodide. Biochemistry Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays Miguel A. Betancourt-Solis1, James A. McNew1 1Department of BioSciences, Rice University Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity. Genetics Designing Automated, High-throughput, Continuous Cell Growth Experiments Using eVOLVER Zachary J. Heins1,2, Christopher P. Mancuso1,2, Szilvia Kiriakov1,3, Brandon G. Wong1,2, Caleb J. Bashor4, Ahmad S. Khalil1,2,5 1Biological Design Center, Boston University, 2Department of Biomedical Engineering, Boston University, 3Program in Molecular Biology, Cell Biology and Biochemistry, Boston University, 4Department of Bioengineering, Rice University, 5Wyss Institute for Biologically Inspired Engineering, Harvard University The eVOLVER framework enables high-throughput continuous microbial culture with high resolution and dynamic control over experimental parameters. This protocol demonstrates how to apply the system to conduct a complex fitness experiment, guiding users on programming automated control over many individual cultures, measuring, collecting, and interacting with experimental data in real-time. Immunology and Infection A High-throughput, High-content, Liquid-based C. elegans Pathosystem Quinton L. Anderson1, Alexey V. Revtovich1, Natalia V. Kirienko1 1Department of Biosciences, Rice University Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery. Behavior A Burrowing/Tunneling Assay for Detection of Hypoxia in Drosophila melanogaster Larvae Karen M. Qiang1,2, Fanli Zhou1, Kathleen M. Beckingham1 1Department of Biosciences, Rice University, 2Yale Medical School The protocol describes a simple assay to identify Drosophila melanogaster larvae that are experiencing hypoxia under normal atmospheric oxygen levels. This protocol allows hypoxic larvae to be distinguished from other mutants that show overlapping phenotypes such as sluggishness or slow growth. Environment Microfluidic Devices for Characterizing Pore-scale Event Processes in Porous Media for Oil Recovery Applications Eric D. Vavra*1, Yongchao Zeng*1, Siyang Xiao*1, George J. Hirasaki1, Sibani L. Biswal1 1Department of Chemical and Biomolecular Engineering, Rice University The goal of this procedure is to easily and rapidly produce a microfluidic device with customizable geometry and resistance to swelling by organic fluids for oil recovery studies. A polydimethylsiloxane mold is first generated, and then used to cast the epoxy-based device. A representative displacement study is reported. Developmental Biology Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells Kathryn E. Ware1, Shivee Gilja1, Shenghan Xu1, Samantha Shetler1, Mohit K. Jolly2, Xueyang Wang3, Suzanne Bartholf Dewitt4, Alexander J. Hish1, Sarah Jordan1, William Eward5, Herbert Levine2, Andrew J. Armstrong4, Jason A. Somarelli1 1Department of Medicine, Duke University, 2Department of Bioengineering, Rice University, 3Department of Molecular Genetics and Microbiology, Duke University, 4Solid Tumor Program and the Duke Prostate Center, Duke University Medical Center, 5Duke University Medical Center We present here a cell culture method for inducing mesenchymal-epithelial transitions (MET) in sarcoma cells based on combined ectopic expression of microRNA-200 family members and grainyhead-like 2 (GRHL2). This method is suitable for better understanding the biological impact of phenotypic plasticity on cancer aggressiveness and treatments. Medicine Testing the Efficacy of Pharmacological Agents in a Pericardial Target Delivery Model in the Swine Tinen L. Iles1, Brian Howard2, Stephen Howard3, Stephen Quallich2, Christopher Rolfes2, Eric Richardson4, Hanna R. Iaizzo5, Paul A. Iaizzo1 1Surgery, University of Minnesota, 2Biomedical Engineering, University of Minnesota, 3Medtronic, Inc., 4Bioengineering, Rice University, 5Pharmacy, University of Wisconsin We have developed a swine model for the target delivery of pharmacological agents within the pericardial space/fluid. Using this approach, the relative benefits of administered agents on induced atrial fibrillation, relative refractory periods and/or ischemic protection can be investigated. Bioengineering Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites Hojin Kim1, Bohan Zhu1, Huiying Chen2, Oluwatomiyin Adetiba2, Aditya Agrawal1, Pulickel Ajayan3, Jeffrey G. Jacot2,4, Rafael Verduzco1,3 1Chemical and Biomolecular Engineering, Rice University, 2Bioengineering, Rice University, 3Materials Sciences and NanoEngineering, Rice University, 4Congenital Heart Surgery Services, Texas Children’s Hospital We demonstrate the preparation of siloxane-based and epoxy-based liquid crystal elastomers (LCEs) and LCE nanocomposites. The LCEs are characterized with respect to reversible strain, liquid crystal ordering, and stiffness. As a potential application, we demonstrate their use as shape-responsive substrates in a custom device for active cell culture. Biology Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control Zachary A. Crannell*1, Brittany Rohrman*1, Rebecca Richards-Kortum1 1Department of Bioengineering, Rice University Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data. Bioengineering Diagnosis of Neoplasia in Barrett’s Esophagus using Vital-dye Enhanced Fluorescence Imaging Daniel P. Perl1, Neil Parikh1, Shannon Chang1, Paul Peng1, Nadhi Thekkek3, Michelle H. Lee1, Alexandros D. Polydorides2, Josephine Mitcham1, Rebecca Richards-Kortum3, Sharmila Anandasabapathy1 1Department of Gastroenterology, Icahn School of Medicine at Mount Sinai, 2Department of Pathology, Icahn School of Medicine at Mount Sinai, 3Department of Bioengineering, Rice University Vital-dye enhanced fluorescence imaging (VFI) is a novel in vivo technique that combines high-resolution epithelial imaging with exogenous topical fluorescent contrast to highlight glandular morphology and delineate neoplasia (high grade dysplasia and cancer) in the distal esophagus. Medicine Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy Stuart J. Corr1,2, Brandon T. Cisneros1,2, Leila Green1, Mustafa Raoof1, Steven A. Curley1,3 1Department of Surgical Oncology, University of Texas M.D. Anderson Cancer Center, 2Department of Chemistry, Rice University, 3Mechanical Engineering and Materials Science, Rice University We describe the protocols used to investigate the interactions of 13.56 MHz radiofrequency (RF) electric-fields with gold nanoparticle colloids in both non-biological and biological systems (in vitro/vivo). These interactions are being investigated for applications in cancer therapy. Immunology and Infection Mass Spectrometric Analysis of Glycosphingolipid Antigens Alexandra Bili Yin1, David Hawke2, Dapeng Zhou3,4 1Undergraduate Program, Rice University, 2Proteomics Facility, Department of Pathology, University of Texas MD Anderson Cancer Center, 3Department of Melanoma Medical Oncology, University of Texas MD Anderson Cancer Center, 4University of Texas Graduate School of Biological Sciences at Houston A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards. Engineering Terahertz Microfluidic Sensing Using a Parallel-plate Waveguide Sensor Victoria Astley1, Kimberly Reichel1, Rajind Mendis1, Daniel M. Mittleman1 1Department of Electrical and Computer Engineering, Rice University The procedure for implementing a refractive index sensor for terahertz frequencies based on a grooved parallel-plate waveguide geometry is described here. The method yields a measurement of the refractive index of a small volume of liquid through monitoring of the shift in the resonant frequency of the waveguide structure Neuroscience Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation Shannon L. Griswold1, Peter Y. Lwigale1 1Department of Biochemistry and Cell Biology, Rice University An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo. Bioengineering High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging Mark Pierce1, Dihua Yu2, Rebecca Richards-Kortum1 1Department of Bioengineering, Rice University, 2Department of Molecular and Cellular Oncology, The Univeristy of Texas M. D. Anderson Cancer Center In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.