Rapid microfluidic multiplexing of proteins for deciphering spatial organelle networks
Author(s)
Venkatesan, Mythreye
Advisor(s)
Coskun, Ahmet F.
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Abstract
New technologies in advancing biotechnology and biomedical engineering have shown us that organelles play many roles in human health and disease such as maintaining homeostasis, regulating growth and aging, and generating energy. Organelle diversity in cells not only exists between cell types but also between individual cells. Therefore, studying the distribution of organelles at the single-cell level is important to understand the functioning of cells. This thesis aims to understand the spatial organization of organelles and the interactions between them in mesenchymal stem cells using multiplexed imaging of proteins. Mesenchymal stem cells are multipotent cells that have been explored as a therapeutic for treating a variety of diseases including myocardial infarction and traumatic brain injury. Studying how organelles are structured in these cells can answer questions about their function and potential. In this work, rapid cyclic immunofluorescence was performed to decipher the subcellular localization of ten organelle markers in the bone marrow and umbilical cord stem cells. Spatial correlations, colocalization analysis, and clustering were carried out on the 10-plex data to explore relations between the organelles and compare between the two cell types. An automated microfluidic system was also developed to handle the repetitive processes, for instance, relabeling and removing signals from the sample, during rapid cyclic immunofluorescence. This system will advance the multiplexed protein imaging method to provide rapid experiments and imaging on the same microscope platform. In the future, a data-driven single-cell approach provided by automated and rapid subcellular proteomic imaging can enable personalized stem cell therapeutics.
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Date
2021-08-30
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Thesis