Automation of Microraft Arrays for Stem Cell Analysis and Sorting Public Deposited

Downloadable Content

Download PDF
Last Modified
  • March 20, 2019
Creator
  • DiSalvo, Matthew
    • Affiliation: School of Medicine, UNC/NCSU Joint Department of Biomedical Engineering
Abstract
  • Induced pluripotent stem cells (iPSCs) are reprogrammed somatic cells with the potential to revolutionize personalized medicine, disease modeling, and tissue engineering. Emerging therapies based upon human iPSCs (hiPSCs) are already under development for degenerative diseases such as age-related macular degeneration (AMD). Despite the ready availability of hiPSCs, their enormous clinical and research potential is limited by the need to purify the cells during generation, genetic editing, and differentiation using tedious manual methodologies. This dissertation describes the automation and further development of microraft arrays to perform the isolation and splitting of hiPSCs colonies, which is the primary bottleneck in hiPSC purification pipelines. Microraft arrays are elastomeric microwell arrays with releasable magnetic cell culture and transfer elements, or “microrafts,” held within each microwell. Microraft arrays enable the identification of cells by imaging cytometry and the isolation of cells and their associated microrafts by dislodgement from the microarray, followed by magnetic manipulation into secondary vessels. The microraft array platform has been previously shown to be automatable and able to sort cells with exceptional viability and efficiencies. However, previous platforms have lacked the speed and robustness to perform large-scale microraft releases. Furthermore, previous microraft array designs were not designed to culture isolated microcolonies of hiPSCs. In this dissertation, microraft arrays were redesigned to isolate hundreds of microcolonies of cells, each within a nested grid of microrafts. Novel microarray microfabrication and computational modeling methods were developed to enable automated and robust imaging of microraft arrays. Image acquisition and analysis software was created to perform label-free detection of hiPSC microcolonies on microraft arrays and, in a separate application, to monitor colonic organoids. Additionally, a high-throughput automated microraft release and collection platform was developed that, for the first time, made used of real-time imaging to intelligently maximize the robustness and speed of microraft releases. This platform was utilized to isolate, culture, monitor, and successfully split hundreds of hiPSC microcolonies, thus demonstrating its utility for hiPSC purification.
Date of publication
Keyword
DOI
Resource type
Rights statement
  • In Copyright
Advisor
  • Cowley, Dale
  • Lalush, David
  • Ramsey, John
  • Allbritton, Nancy
  • Ligler, Frances
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2018
Language
Parents:

This work has no parents.

Items