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. 2021 Dec 14;9:796960. doi: 10.3389/fcell.2021.796960

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Copyright © 2021 Li, Walsh, Truong, Petersen, Dutton and Hubel.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Effects of undercooling on freezing behaviors of D5 crest, D7 SN, and D14 mSN, respectively, as observed by low-temperature Raman spectroscopy, comparing different ice nucleation temperatures between −4°C and −8°C. Data are represented as mean ± 95% confidence interval. n = 10, except for striped columns representing measurements of subset population n = 5 for D7 SN and n = 7 for D14 mSN due to interference of intracellular ice. n-D, non-DMSO; D, DMSO. n.s.: p > 0.05; *p < 0.05 (see Supplementary Figure S2 for method illustration of each metric.). (A) Intracellular ice formation (blue) significantly increased with greater undercooling only in the case of D7 SN in the DMSO solution and D14 mSN in both non-DMSO and DMSO solutions; no significant change otherwise. (B) Membrane partitioning of non-DMSO CPA significantly decreased in D14 mSN subject to greater undercooling, partitioning of DMSO lower than that of non-DMSO CPA across all three cell stages. (C) Cellular integrity in terms of spatial autocorrelation of the amide I signal of protein decreased significantly for D14 mSN with greater undercooling and decreased further in the DMSO solution; no significant loss of integrity in D5 crest or D7 SN. (D) Significant cytochrome C (cyt C) release was observed for D14 mSN in the DMSO solution but not for any cell stage in the non-DMSO CPA. Kruskal–Wallis ANOVA performed for non-normal distribution of cyt C Moran’s I values.