Freezing Responses in DMSO-Based Cryopreservation of Human iPS Cells: Aggregates Versus Single Cells

Rui Li, Guanglin Yu, Samira M. Azarin, Allison Hubel

Research output: Contribution to journalArticlepeer-review

27 Scopus citations


Inadequate preservation methods of human induced pluripotent stem cells (hiPSCs) have impeded efficient reestablishment of cell culture after the freeze-thaw process. In this study, we examined roles of the cooling rate, seeding temperature, and difference between cell aggregates (3-50 cells) and single cells in controlled rate freezing of hiPSCs. Intracellular ice formation (IIF), post-thaw membrane integrity, cell attachment, apoptosis, and cytoskeleton organization were evaluated to understand the different freezing responses between hiPSC single cells and aggregates, among cooling rates of 1, 3, and 10°C/min, and between seeding temperatures of -4°C and -8°C. Raman spectroscopy images of ice showed that a lower seeding temperature (-8°C) did not affect IIF in single cells, but significantly increased IIF in aggregates, suggesting higher sensitivity of aggregates to supercooling. In the absence of IIF, Raman images showed greater variation of dimethyl sulfoxide concentration across aggregates than single cells, suggesting cryoprotectant transport limitations in aggregates. The ability of cryopreserved aggregates to attach to culture substrates did not correlate with membrane integrity for the wide range of freezing parameters, indicating inadequacy of using only membrane integrity-based optimization metrics. Lower cooling rates (1 and 3°C/min) combined with higher seeding temperature (-4°C) were better at preventing IIF and preserving cell function than a higher cooling rate (10°C/min) or lower seeding temperature (-8°C), proving the seeding temperature range of -7°C to -12°C from literature to be suboptimal. Unique f-actin cytoskeletal organization into a honeycomb-like pattern was observed in postpassage and post-thaw colonies and correlated with successful reestablishment of cell culture.

Original languageEnglish (US)
Pages (from-to)289-299
Number of pages11
JournalTissue Engineering - Part C: Methods
Issue number5
StatePublished - May 2018

Bibliographical note

Funding Information:
This work was funded by R01EB023880. Parts of this work were carried out in the Characterization Facility, University of Minnesota, which receives partial support from NSF through the MRSEC program. The authors thank their colleagues at Ogle laboratory in the Department of Biomedical Engineering for providing the flow cytometry system.

Publisher Copyright:
© Copyright 2018, Mary Ann Liebert, Inc.


  • Raman spectroscopy
  • cryopreservation
  • dimethyl sulfoxide
  • induced pluripotent stem cells


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