Cryopreservation of Stem Cells: Evaluating Approaches for Clinical Potential

Posted on Updated on

Despite a growing number of publications discussing various aspects of stem cell cryopreservation, a clear understanding of an optimal set of guidelines, including protocols and, most importantly, cryopreservation solutions that are clinically-suitable is still lacking. The unpredictable behavior and differing biophysical characteristics of different cell types has made making such generalizations less straightforward, as an understanding of individualized cell behavior is becoming of increasing importance.

A recent study by the University of Leuven in Belgium sought to bring some more clarity to these issues. The authors compared seven different freezing and thawing protocols using human amniotic fluid-derived stem cells.

These were:

  • (1) 10% dimethyl sulfoxide (DMSO)
  • (2) 2.5% DMSO, caspase inhibitor, and catalase
  • (3) 5% glycerol, caspase inhibitor, and catalase
  • (4) sperm freezing medium
  • (5) slow-freezing solution
  • (6) ethylene glycol, sucrose, and Ficoll 70
  • (7) vitrification solution

Medium 4, sperm freezing medium, was Irvine Scientific’s TYB Freezing Medium, solution 6 was Vitrolife’s FreezeKit medium, while the vitrification solution (7) was Vitrolife’s RapidVit vitrification kit.

While protocols 1, 2, 5 and 6 resulted in successful recovery of hAFSCs based on live/dead assay, a lower CD marker expression profile was much weaker for protocol 2.

Expression levels of GAPDH, Oct-4, SOX17, vimentin, KSP and NCAM showed increased SOX17 gene expression for protocols 1, 2 and 6 compared to the unfrozen control samples.

Taking all of the results into account, the authors identified approaches 1, 5 and 6 as being superior in terms of recovery of cells by yielding a significant amount of cells with strong surface marker expression.

Screen Shot 2015-02-15 at 6.18.18 PM


Out of these, the slow-freezing solution (5) was identified by the authors as being the most robust in terms of cell recovery and desirable properties after thawing, and recommendations were made as to its clinical use.

While these are preliminary results that apply to stem cells derived from amniotic fluids, the paper raises important points about the development of freezing solutions by highlighting the important fact that cell-specific behavior does not necessarily show consistency across a range of assays when analyzing treatment response and that multiple analyses need to be considered as a whole.

At Akron, we have been investigating a range of new solutions for cell cryopreservation that are both DMSO-based as well as DMSO-free and have developed a strong know-how of products and solutions for various cell types, particularly those that are free from DMSO. If you have any questions or need help understanding your options for cell cryopreservation (without or with DMSO), feel free to get in touch with us via email at

4 thoughts on “Cryopreservation of Stem Cells: Evaluating Approaches for Clinical Potential

    Samad said:
    February 16, 2015 at 6:29 am

    Needless to say that, cryopreservation is a small part of the process of producing stem cells and their derivatives for therapy. But the unpredictable behavior and differing biophysical characteristics of different cell types has made making such generalizations less straightforward- I agree to this point.

    Henry E. Young, PhD said:
    February 17, 2015 at 7:36 pm

    We found that freezing temperature played a large part in the cryopreservation process dependent on cell type. For example, differentiated cells and progenitor cells preferred being flash frozen in liquid nitrogen (>95% recovery). In contrast, mesodermal stem cells preferred cryopreservation at -70C (>98% recovery), while adult-derived pluripotent stem cells and totipotent stem cells preferred cryopreservation at -80C (>98% recovery). We also determined (empirically) that the percentage as well as purity of the DMSO that was utilized mattered significantly. We utilized 7.5% v/v ultra-pure DMSO (i.e., 99.99% pure) with slow freezing and flash thawing to achieve the successes noted above.

    Henry E. Young, PhD said:
    February 17, 2015 at 7:40 pm

    Oh, and I might also add that we utilized differentiated cells, progenitor cells, mesodermal stem cells, pluripotent stem cells, and totipotent stem cells derived from 37 different organs and tissues from 3 different mammalian species (mouse, rat, and human) for our studies. We also tested in the above cell types derived from avian (Gallus domesticatus) and the results were the same.

    Miho K Furue PhD., DDS. said:
    February 18, 2015 at 6:59 pm

    There is not any evidence that the cells can stably preserve their phenotypes in new freezing medium for more than 10 years. Thus, it is suggested that although these new methods would be worth to be challenged, the conventional cryopreservation should be backed up as a precaution. We have uploaded an improved protocol using DMSO at Protocol Exchange (2014) doi:10.1038/protex.2014.012; A simple improvement of the conventional cryopreservation for human ES and iPS cells). We thaw the frozen cells by warm medium directly and suspend the cells in warm medium. This method increase AP-positive hESC/hiPSC colony numbers.

Leave a Reply

Fill in your details below or click an icon to log in: Logo

You are commenting using your account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s