A study on these freezing responses, relating to aggregates versus single cells, has just been published in Tissue Engineering (on-line February 2018) by Rui Li, Guanglin Yu (pictured here) and others working with Prof Allison Hubel's team at the Department of Mechanical Engineering University of Minnesota. Human induced pluripotent stem cells (hiPSCs) are multicellular aggregates attracting much interest in tissue engineering, disease modelling and personalised medicine. They can be frozen either as aggregates or single cells depending upon the application. For both clinical and scientific purposes, effective cryopreservation of hiPSCs is required for transportation, storage of frozen hiPSCs and other downstream uses. However, cryopreserved hiPSCs are vulnerable to the loss of viability, function or pluripotency.

It is known that inadequate preservation methods of hiPSCs have impeded efficient re-establishment of cell culture after their freeze-thaw. In the study the roles of cooling rate, seeding temperature and the difference between cell aggregates and single cells in controlled rate freezing were examined using, inter alia, Raman spectroscopy, as a tool for understanding cell responses to the freezing environment. The Raman spectroscopy was used to observe both hiPSC single cells and aggregates frozen at three cooling rates and two seeding temperatures; it suggested higher sensitivity of aggregates to supercooling than previously thought. The work will deepen understanding of behaviours of single cells and aggregates frozen at various conditions and help promote the development of improved cryopreservation protocols for human induced pluripotent stem cells.

For single cells, slow cooling rates allowed significantly better preservation of membrane integrity than higher cooling rate (10˚C/min) regardless of the seeding temperature. For aggregates, however, slow cooling rates (1, 3˚C/min) combined with high seeding temperature (4˚C) had little effect on the membrane integrity but resulted in significantly better cell attachment than higher cooling rate (10˚C/min) or low seeding temperature (8˚C). The authors say there are advantages of using a seeding temperature of 4˚C compared to 8˚C suggesting that the range of seeding temperatures of 7˚C to 12˚C quoted in much literature may be sub-optimal, and that seeding temperature should be considered as a critical parameter when designing cryopreservation protocol for hiPSCs. Guanglin Yu, see photo above, carried out a lot of the experimentation. He said "...we used manual seeding for nucleation of the sample. We sprayed the sample with a narrow stream of liquid nitrogen. We saw different cell responses of HiPSC with seeding temperature of -4˚C and -8˚C using Raman microscopy. This made it clear that we wanted to control the temperature at which ice formed in the extracellular space for controlled rate freezing experiments."

The paper indicates that hiPSCs respond to freezing in very complex fashion, and successful establishment of post thaw culture depends on various critical factors. Further studies will need to not only continue exploring additional factors to optimise the freezing protocol for hiPSCs but investigate the biological pathways connecting the factors and the observed cryopreservation outcomes to provide targets for future development of cryoprotectants.

For further information
Contact Prof Hubel's Lab: This email address is being protected from spambots. You need JavaScript enabled to view it.
Tissue Engineering: https://www.liebertpub.com/doi/abs/10.1089/ten.TEC.2017.0531
Planer Controlled Rate Freezer: https://planer.com/products/cryo-freezers/medium-crf.html

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