Description
- Abstract:
- Sorting methods are important tools that enable cellular subpopulation isolation for basic science investigations and clinical therapies. Each method can be characterized by a set of parameters including resultant cell viability, specificity, yield, and throughput, among others. While the specificities of common sorting methods are often discussed, yields, throughputs, and isolated cell viabilities are rarely reported. However, a holistic, quantitative understanding of these parameters is required for making appropriate sorting method decisions. The goals of this thesis were to quantify these important sorting efficiency parameters in the two most common cell sorting methods, fluorescence- and magnetic-activated cell sorting (FACS and MACS, respectively), and to develop a novel sorting method to address observed shortcomings. To test FACS and MACS, we used each method to separate mixtures of osteogenically primed stromal vascular fraction (SVF) and A375 human melanoma cells based on alkaline phosphatase liver/bone/kidney (ALPL) protein expression. Using suggested antibody and microbead concentrations, MACS delivered high-yield isolations but inaccurate ALPL+/ALPL- splits compared to FACS. Rigorous optimization of antibody and microbead concentrations enabled MACS to accurately separate ALPL+ and ALPL- cells with high yield. Additionally, MACS-isolated subpopulations displayed statistically higher viability than those isolated by FACS. Despite enabling high-yield, high-viability isolations, MACS was not capable of non-binary separations. As such, we set out to design a particle-based sorting method that would combine the gentle, bulk processing of MACS with the non-binary separation afforded by FACS. By labeling cells with small, massive particles targeting a protein of interest prior to density gradient centrifugation, we hypothesized we could achieve non-binary separation. This mass-added density centrifugation (MADC) enabled the binary separation of mixtures of bead-treated and untreated A375s using continuous Percoll gradients. However, neither binary nor non-binary MADC separations of MeWo human melanoma cells were successful, likely due to widespread cellular aggregation. The results presented in this thesis will provide researchers with a thorough characterization of MACS and FACS, a MACS optimization template, and an exciting alternative sorting method, MADC.
- Notes:
- Thesis (Ph. D.)--Brown University, 2019
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Citation
Sutermaster, Bryan,
"Investigations in Cell Sorting Techniques"
(2019).
Biomedical Engineering Theses and Dissertations.
Brown Digital Repository. Brown University Library.
https://doi.org/10.26300/v6e3-2230
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Biomedical Engineering Theses and Dissertations
Theses and Dissertations for the Biomedical Engineering department....