Graduation Year

2011

Document Type

Thesis

Degree

M.S.B.E.

Degree Granting Department

Chemical Engineering

Major Professor

Mark J. Jaroszeski, Ph.D.

Co-Major Professor

Don F. Cameron, Ph.D.

Committee Member

Nathan D. Gallant, Ph.D.

Committee Member

Richard J. Connolly, Ph.D.

Keywords

Allograft, Electropermeabilization, Fusogenic, Immunosuppresion, Xenograft

Abstract

Cell transplantation therapy is a potentially powerful tool and can be used to replace defective cells with healthy cells. This offers the possibility of alleviating the destructive symptoms for many diseases such as Parkinson's disease, Alzheimer's disease, stroke, spinal cord trauma, Type I diabetes and many more. While there are many diseases that could be positively impacted from cell transplantation therapy, the focus of this research is insulin dependent, Type I Diabetes.

The Islets of Langerhans are composed of various types of cells located in the pancreas and are responsible for a variety of biochemical functions. Specifically, the beta Islet cells are responsible for production of the hormone insulin that regulates and aids in biosynthesis of glucose. Transplantation of isolated allografted pancreatic islets, which contain insulin producing cells, into diabetic rats has proven to be highly successful. However, these transplantations involve using medications for long term immunosuppression to defend against an undesired host immune response. Immunosuppressive medications are both costly and illicit additional side effects that can be detrimental to the host. This research focuses on the use of testicular derived Sertoli cells that have been publicized to provide localized immunoprotection.

Electrofusion is a process that can be used to fuse homogeneous and heterogeneous cell types by promoting the creation of micropores in the cell's lipid bilayer. This renders the cell temporarily fusogenic, or capable of facilitating fusion. Cells must then be brought into contact with one another via mechanical, chemical or viral means. This research study proposes to optimize electrofusion technology to create novel, secretory hybrids composed of Islet and Sertoli cells that are immunoprotected and produce insulin in response to a glucose challenge.

The components of the electrofusion device include a Sterlitech 0.2 ìm microporous membrane, a woven cellulose absorbent pad, two aluminum electrodes and a chamber body and top injection molded using Delrin. Preliminary experiments using B16-F10 murine melanoma cells incorporated with centrifugation to increase cell to cell contact resulted in an average fusion yield of 18.9% ± 8.1 SD using a field strength of 2500 V/cm, 8 pulses and a 250 ìs pulse length. Additionally, lab synthesized electroporation buffers containing 8.5% sucrose (w/v) and 0.3% glucose increased total and viable fusion yields to 37.1% ± 9.3 SD and 13.8% ± 2.1 SD, respectively. These results showed promise and should be further validated with additional cell lines and tissues to corroborate reproducibility.

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