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Membrane Sandwich Electroporation for In Vitro Gene Delivery

Fei, Zhengzheng
http://rave.ohiolink.edu/etdc/view?acc_num=osu1251384862

Abstract Details

2009, Doctor of Philosophy, Ohio State University, Chemical Engineering.
Gene therapy is the delivery of therapeutic genes into cells and tissues with the aim of treating and curing a disease. As an enhanced understanding of the roles of genes in health and disease, gene therapy is showing promise against various diseases such as cancer, diabetes, Parkinson's disease, and several inherited physiological defects. Viral transduction is very efficient, but safety issues, such as immune and inflammatory responses, have hampered their clinical uses in humans. Non-viral methods, including either chemical transfection with cationic lipids/polymers or physical transfection using electroporation/microinjection, are becoming attractive approaches. Electroporation is one of the most popular non-viral gene transfer methods for in vitro cell transfection. Initial studies with electroporation experienced very low transfection efficiencies and cell viability, severely limiting the development of this technology. The emergence of nucleofection (a modified electroporation technology) provided an efficient means for transfecting cells in vitro. However, nucleofection still encounters many limitations such as the large number of cells required and high cost involved. Moreover, cell viability is still an issue due to the high electric voltage used and the non-uniform electric field strength distribution generated during the process. To address these problems, we propose to develop an electroporation system based on an innovative micro-/nanoengineering technology for in vitro gene delivery. In our approach, electroporation is carried out in a mild and uniform electric field, with potential for a wider process window that can be generated to cover a wide range of cell lines and even primary cells. A new membrane sandwich electroporation (MSE) approach was demonstrated using plasmids GFP and SEAP as model materials. NIH 3T3 fibroblasts were tested and a significant improvement in transgene expression was observed compared to current electroporation techniques. In the MSE method, the focused electric field enhances cell permeabilization at a low electric voltage, leading to high cell viability; more important, the sandwich membrane configuration is able to provide better gene confinement near the cell surface, facilitating gene delivery into the cells. Next, we demonstrated the use of femtosecond laser fabricated micro-nozzle arrays on a gelatin-coated PET membrane for MSE. Using micro-nozzle array enhanced MSE, we observed high and uniform gene transfection, and good cell viability of mouse embryonic stem (ES) cells compared to the bulk electroporation. Since typically cells or tissues from the patients are very limited and therapeutic materials such as plasmids and oligonucleotides are very expensive, the ability to treat a small number of cells (i.e. a hundred) offers great potential to work with hard-to-harvest patient cells for patient-specific ex vivo gene therapy and in vitro pharmaceutical kinetic studies. Numerical calculation of transmembrane potential qualitatively explains the observed differences between MSE and bulk electroporation. Since there’s a good correlation between transfection results and transmembrane potential calculations, the simulation process with the threshold experiments can be used to predict the transfection results, and thus largely reduced the trial-and-error window size. Furthermore, we successfully integrated an electrospun nanofiber scaffold as a cell-binding substrate into MSE, called nanofiber based MSE. With a micro-well spacer, the uniform size of mouse ES cell colonies were obtained, and plasmid transfection by electroporation were performed during colony formation. In addition, repeated plasmid SEAP transfection of NIH 3T3 fibroblasts was tested and better cell survival and recovery rate was observed using the electrospun nanofiber scaffold as compared to using micro-porous membrane. Due to its capacity of extend the exposure time with reprogramming factors, nanofiber based MSE demonstrated the potential for efficient induced pluripotent stem (iPS) cell generation by repeated plasmid transfection.
Ly James Lee (Advisor)
electroporation; gene delivery; cell culture; stem cells

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Citations

  • Fei, Z. (2009). Membrane Sandwich Electroporation for In Vitro Gene Delivery [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1251384862

    APA Style (7th edition)

  • Fei, Zhengzheng. Membrane Sandwich Electroporation for In Vitro Gene Delivery. 2009. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1251384862.

    MLA Style (8th edition)

  • Fei, Zhengzheng. "Membrane Sandwich Electroporation for In Vitro Gene Delivery." Doctoral dissertation, Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1251384862

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.