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Dissertation_Junfeng_Shi_20170818.pdf (7.99 MB)

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Development of Nanoelectroporation-based Biochips for Living Cell Interrogation and Extracellular Vesicle Engineering

Shi, Junfeng, Leng
http://rave.ohiolink.edu/etdc/view?acc_num=osu1503059915552435

Abstract Details

2017, Doctor of Philosophy, Ohio State University, Mechanical Engineering.
Delivery of exogenous materials plays a key role a diversity of biomedical and pharmaceutical applications. Precise, rapid and benign introduction of biomolecules into a large population of cells has thus long fascinates the scientific community. To circumvent the safety concern raised by viral vectors, a variety of non-viral delivery approaches have been developed, including chemical carrier-mediated methods and physical membrane-penetrating methods. Although novel synthetic lipoplex and polyplex nanocarriers provide opportunities for targeted delivery both in vivo and in vitro, the delivery process is slow and inefficient -- only a small fraction of encapsulated materials is eventually delivered to cell interior after endocytosis followed by endosomal escape. Electroporation has been the leading physical delivery method since its invention about four decades ago. Bulk electroporation (BEP) is the commercially available electroporation system. While BEP offers the advantage of simplicity to use without any package of delivery materials, it suffers from low cell viability and significant cell-to-cell variation owing to the non-uniform electric field imposed on the cells randomly suspended in the bulk solution. A rapid growth of microfluidics has been witnessed since 2000, as microfabrication techniques get mature. The spatial confinement offers an opportunity to accurately control over fluid transport as well as the electric field distribution. In this work, a miniaturized version of electroporation -- nano-electroporation (NEP) was integrated in three biochip platforms, which could revitalize this classic physical delivery method. Large-scale single-clone-resolution living cell interrogation, including single- cell motility, drug resistance, and underlying molecular mechanisms, based on a 3D NEP- cell migration platform was demonstrated using a clinically relevant study of patient-derive glioma stem cells (GSCs) in the highly lethal brain tumor – glioblastoma. This platform was utilized to follow the dynamics of phenotypic shift of cancer stem cells and thus has potential to shed a new perspective for current solid tumor research and help develop novel therapeutic strategy specifically targeting a subpopulation with high plasticity within cancer stem cells. To enhance the transfection efficiency and the versatility of transfected cell types (i.e., both adherent and suspension cells) in the 3D NEP platform, a scalable microfluidic device enabling rapid, massive and accurate hydrodynamic-based cell trapping critical for large-scale 3D NEP cell transfection was introduced. Various cargoes from large plasmids to small oligos have been shown to be uniformly NEP-delivered into the trapped cell array in a controllable and uniform manner. Such innovative micro-/nano-technology-enabled cell transfection platform could be valuable for cell reprogramming and ex vivo cancer adoptive immunotherapies. For the first time, we reported that NEP can not only serve as a unique gene delivery tool, capable of deterministic and benign non-endocytic transfection, but also be a revolutionary extracellular vesicle (EV) engineering tool promising for exosomal mRNA/miRNA transfer in regenerative medicine and cancer therapy, by orchestrating a heat-shock-mediated cellular responses in NEP transfected cells. An integrated NEP- Tethered Lipoplex Nanoparticles (TLN) biochip was also developed for in situ characterization of exosomal content secreted from NEP-transfected cells at the single- exosome level.
James Lee (Advisor)
209 p.
Mechanical Engineering
Nano-electroporation, Microfluidics, Microfabrication

Recommended Citations

Citations

  • Shi, Leng, J. (2017). Development of Nanoelectroporation-based Biochips for Living Cell Interrogation and Extracellular Vesicle Engineering [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1503059915552435

    APA Style (7th edition)

  • Shi, Leng, Junfeng. Development of Nanoelectroporation-based Biochips for Living Cell Interrogation and Extracellular Vesicle Engineering. 2017. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1503059915552435.

    MLA Style (8th edition)

  • Shi, Leng, Junfeng. "Development of Nanoelectroporation-based Biochips for Living Cell Interrogation and Extracellular Vesicle Engineering." Doctoral dissertation, Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1503059915552435

    Chicago Manual of Style (17th edition)

osu1503059915552435
574
© 2017, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.