Li, Rui2021-10-252021-10-252021-09https://hdl.handle.net/11299/225110University of Minnesota Ph.D. dissertation. September 2021. Major: Biomedical Engineering. Advisor: Allison Hubel. 1 computer file (PDF); xix, 248 pages.Human induced pluripotent stem cells (hiPSCs) possess the potential to differentiate into any cell of the human body for translational research towards drug discovery, disease modeling, cell therapy, and tissue engineering applications. hiPSCs and differentiated cells of certain lineages and maturity have been challenging to cryopreserve. Effective cryopreservation will enable off-the-shelf use of the hiPSC-derived personalized medicine platforms and therapeutic products. Systematic understanding of cryobiological mechanisms is a key to the effective design of cryopreservation methodology. We hypothesize that the mechanism of cryopreservation challenge varies between different forms of the same cell type, particularly multicellular aggregates versus single cells of hiPSCs, as well between different developmental stages of the same tissue-specific lineage derived from hiPSCs; for example, neural crest cells versus immature versus mature sensory neurons. Using low-temperature Raman spectroscopy as a tool for label-free mechanistic discovery in molecular and cell cryobiology, this research demonstrates that multicellular aggregates of hiPSCs are significantly more sensitive to undercooling stresses due to their lower membrane fluidity than single cells. Similarly, undercooling sensitivity of different hiPSC-derived neuronal cell stages also inversely correlate with their respective membrane fluidity. However, a synergistic formulation of non-DMSO osmolyte-based cryoprotective agents (CPAs) can effectively reduce cell sensitivity to undercooling otherwise manifested by DMSO, by strengthening the hydrogen bonds with water and inhibiting intracellular ice. We find that hiPSC-derived developmental stages with larger cell sizes require significantly slower cooling rates than those with smaller cell sizes to avoid intracellular ice and consequent cell damages upon fast cooling. The mechanisms of action of sucrose, glycerol, L-isoleucine, human serum albumin, and poloxamer 188 are respectively examined. Their thermophysical interaction with water, with each other, and biological interaction with cells act in concert to optimize pre-freeze cell stability, post-thaw survival and function, producing cells superior in quality and undercooling tolerability to those cryopreserved in DMSO-based alternative formulations. Learning the mechanisms of cryoinjury in cell forms and cell types that are challenging to cryopreserve, as well as the mechanisms of cryoprotection by different CPAs under different cooling rates and nucleation temperatures, allows us to target identifiable problems with effective solutions. The outcome of this dissertation will bring empirical perspectives into designs of cryopreservation strategy for hiPSCs and hiPSC-derived neural crest cells, sensory neurons and present a potentially imitable approach for future cryobiological studies of other hiPSC-based cell technologies.enThesis or Dissertation