Automation of the Microraft Array Platform for the Selection of Cytotoxic T Lymphocytes

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  • March 20, 2019
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  • Attayek, Peter
    • Affiliation: School of Medicine, UNC/NCSU Joint Department of Biomedical Engineering
Abstract
  • Adoptive cellular therapy (ACT) is an emerging immunotherapy having significant promise in the treatment of a variety diseases, including cancer and chronic infections. ACT involves the infusion of cytotoxic T lymphocytes (CTLs) that recognize viral protein epitopes or, in the case of cancer, a mutated or overexpressed protein epitope present on the surface of diseased cells. While ACT has led to some outstanding clinical results, it is currently very difficult to identify and isolate highly active antigen-specific CTLs, hindering the study and adoption of ACT. The work described in this dissertation is focused on the automation and further development of the microraft array platform for the identification and isolation of CTLs based on cytotoxicity. The microraft array platform consists of an elastomeric microwell array with each individual microwell containing a magnetic, releasable culture site, or microraft. The microraft array platform enables identification of cells or cell events by image cytometry, and the individual microrafts can be released from the array and collected, along with the contents of the microraft. This technology has shown success in sorting single cells and small colonies of cells with high post-sort purity and viability. However, previous implementations of the microraft platform relied on manual identification and isolation of microrafts, limiting the potential of the platform. Furthermore, the microraft array platform was only amenable to adherent cell types which would not enable the isolation of non-adherent CTLs. In this dissertation, the microraft array platform was automated and adapted for use with non-adherent cell types. Gelatin was used to temporarily encapsulate non-adherent cell(s) on a microraft without affecting viability. Image acquisition, processing and analysis software was written to facilitate the automated identification of cell(s) based on a variety of temporal fluorescence-based properties. Additionally, hardware and software were designed and implemented to automate the release and collection of individual microrafts. This automated microraft platform was used to successfully isolate antigen-specific CTLs, demonstrating its future potential for aiding in the study of ACT.
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  • In Copyright
Advisor
  • Allbritton, Nancy
  • Walker, Glenn
  • Taylor, Anne
  • Armistead, Paul
  • Gomez, Shawn
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2016
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