Rapid single-cell electroporation for labeling organotypic cultures

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2010.Vita.Includes bibliographical references (p. 37-39).Single-cell electroporation is a technique for transfecting individual cells in tissue culture at relatively high efficiencies, however it is both time-consuming and low-throughput and this limits the number of different labeling agents that can be effectively introduced into a region of tissue in reasonable periods of time. A novel system that will rapidly load, clean, and accurately position a glass micropipette electrode into tissue culture for single-cell electroporation is proposed. The system will significantly increase the number of different labeling agents that can be introduced into a single tissue culture per unit time. This in turn, will provide a means for improving the study of neural anatomy at cellular resolutions in both tissue culture and in vivo environments.Supported by grants from the National Institutes of Health and by the MIT Dept. of Electrical Engineering and Computer Scienceby Joseph D. Steinmeyer.S.M

Organ-targeted high-throughput in vivo biologics screen identifies materials for RNA delivery

Therapies based on biologics involving delivery of proteins, DNA, and RNA are currently among the most promising approaches. However, although large combinatorial libraries of biologics and delivery vehicles can be readily synthesized, there are currently no means to rapidly characterize them in vivo using animal models. Here, we demonstrate high-throughput in vivo screening of biologics and delivery vehicles by automated delivery into target tissues of small vertebrates with developed organs. Individual zebrafish larvae are automatically oriented and immobilized within hydrogel droplets in an array format using a microfluidic system, and delivery vehicles are automatically microinjected to target organs with high repeatability and precision. We screened a library of lipid-like delivery vehicles for their ability to facilitate the expression of protein-encoding RNAs in the central nervous system. We discovered delivery vehicles that are effective in both larval zebrafish and rats. Our results showed that the in vivo zebrafish model can be significantly more predictive of both false positives and false negatives in mammals than in vitro mammalian cell culture assays. Our screening results also suggest certain structure–activity relationships, which can potentially be applied to design novel delivery vehicles.National Institutes of Health (U.S.) (Transformative Research Award R01 NS073127)National Institutes of Health (U.S.) (Director's Innovator Award DP2 OD002989)David & Lucile Packard Foundation (Award in Science and Engineering)Sanofi Aventis (Firm)Foxconn International Holdings Ltd.Hertz Foundation (Fellowship)University Grants Committee (Hong Kong, China) (Early Career Award 125012)National Natural Science Foundation (China) (81201164)ITC (ITS/376/13)Chinese University of Hong Kong (Grant 9610215)Chinese University of Hong Kong (Grant 7200269

Automation of single-cell techniques in neural tissue

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2014.Cataloged from PDF version of thesis.Includes bibliographical references (pages 125-140).The highly heterogeneous nature of cells in the context of native tissue environments necessitates the development of tools and techniques that can manipulate and analyze samples with single-cell resolution. While the past decades have seen significant progress in analyzing individual cells in tissue, both electrically and morphologically, the ability to genetically manipulate and biochemically analyze such cells in a high-throughput manner has seen only limited advances, and therefore a significant technological gap in accessing cells with single-cell specificity in tissue remains. We present a system design and workflow that fills in this gal) in technology through the implementation of precision automation and redesign of standard biological techniques, resulting in greatly improved throughput while maintaining single-cell accuracy and precision. This thesis comprises three parts: First we discuss the design and implementation of an expandable computer-controlled automation system enabling the rapid maneuvering and targeting of inicropipettes within tissue environments as well as a methodology for cleaning and reuse of these micropipettes to enable significant gains in throughput. Second we apply this automation to transfecting neurons in brain slices with DNA and RNA for subsequent analysis with greater throughput than previous methods. Third, we apply our automation to collecting the contents of single neurons embedded in relevant tissue environments for molecular analysis. The work presented greatly improves the throughput of traditional single-cell methods of transfection and cell-sampling by between one and two orders of magnitude and fills in a gap in the workflow of the rapidly expanding field of single-cell analysis.by Joseph D. Steinmeyer.Ph. D