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

Tissue Engineering. Part C, Methods
Rui LiA Hubel

Abstract

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 parameter...Continue Reading

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Citations

Nov 7, 2019·Tissue Engineering. Part C, Methods·Yuzhao SunSophie X Deng
Feb 11, 2020·Frontiers in Bioengineering and Biotechnology·Rui LiAllison Hubel
Jan 1, 2019·Regenerative Engineering and Translational Medicine·Guanglin Yu, Allison Hubel
Oct 20, 2018·Langmuir : the ACS Journal of Surfaces and Colloids·Irena KratochvílováMartin Falk
Nov 7, 2018·Langmuir : the ACS Journal of Surfaces and Colloids·Guanglin YuAllison Hubel
Aug 26, 2020·The Journal of Physical Chemistry Letters·Kankan QinFrancisco M Fernandes

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Methods Mentioned

BETA
Raman
Flow cytometry

Software Mentioned

FIJI ImageJ

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