Effect of Freeze/Thaw on Gene Expression of Mesenchymal Stromal Cells

Mesenchymal stromal cells, stained with haematoxylin.
Image Source: Wikimedia. Uploaded by OlgaMaslova, under Creative Commons License CC BY-SA 4.0.

With the help of BioCision’s CoolCell® cell freezing containers, scientists explore the effect of freeze/thaw on the global gene expression of mesenchymal stromal cells – and find that surprisingly few things change. That is great news for stem cell therapy enthusiasts.

Mesenchymal stromal cells (MSCs) represent one of the medical community’s greatest hopes for healing hitherto uncurable diseases. These stem cells have the capability to develop into bone, cartilage, muscle and fat cells. They also have immuno-modulatory capacities and can suppress the host’s local immune response – a remarkable feat that makes these cells very attractive for use in transplantation procedures. However, there’s a catch: while mesenchymal stromal cells tend to perform stellarly in tissue culture and pre-clinical experiments, results from human studies remain a mixed bag.

Naturally, scientists are wondering why. Are cryopreservation procedures to blame for the difference? A number of studies recently addressed this issue. Scientists explored whether freeze-thawing affected cell viability, surface antigen profiles and differentiation potential, but no differences in these characteristics were seen between fresh and cryopreserved MSCs. So what is going on?

A group of researchers led by a team of Dutch scientists at the Erasmus Medical Center in Rotterdam turned their attention to gene expression. Could it be that mesenchymal stromal cells that had undergone freeze-thawing were generating a gene expression profile that was very different from that of fresh cells? Their report provided a clear answer. [1] That answer, in brief, was no.

Since fat contains the most resilient non-hematopoietic stem cells in the human body, the scientists used human mesenchymal stromal cells harvested from adipose tissue of healthy volunteers in their experiments. They meticulously studied the gene expression profiles from three different cell lots: (1), fresh MSCs used immediately after harvest, isolation and passage in culture media; (2), cryopreserved MSCs, which had undergone a slow controlled freezing procedure in BioCision’s CoolCell® cell freezing containers and had been thawed subsequently at 37°C; and (3), MSCs harvested from lung tissues of mice 2 hours after delivery into the tail vein of the animals. BioCision’s CoolCell® containers were used for the second cell lot, because they ensured consistency and the highest viability of the mesenchymal stromal cell population after freezing.

Turns out that the profiles between fresh and cryopreserved MSCs did not differ much. In fact, the profiles of fresh and cryopreserved MSCs from the same donor were more similar to each other than those of fresh MSCs that were obtained from different donors. Less than 300 genes were differently expressed, up to only 10fold. Compare that to the over 2,000 genes that were up to 720fold differently expressed between fresh mesenchymal stem cells and those located in murine lung tissue!

Expression of MSC surface markers remained unchanged. Immuno-modulatory capabilities, measured by cytokine and chemokine levels in murine lung tissue after injection of fresh or freeze-thawed mesenchymal stem cells, stayed the same. The only small functional difference the researchers were able to detect, after much searching, was a marginally faster blood clotting time of host blood when exposed to the cryopreserved cells. The scientists attributed this effect to the slightly higher membrane permeability of freeze-thawed MSCs.

Overall, the authors conclude, that “…freeze-thawing procedures have little impact on MSC gene expression…” That is great news, as it supports the concept that stem cells can be cryopreserved and utilized at a later date, simplifying therapy design. Alas, the scientists are left to speculate why some clinical trials with mesenchymal stromal cells did not deliver the expected results. Cryopreservation, if performed correctly, does not seem to be the culprit. So what is? The search continues.


[1] Hoogduijn M.J. et al. Effects of freeze-thawing and intravenous infusion on mesenchymal stromal cell gene expression. Stem Cells Dev February 2016, ahead of print. doi:10.1089/scd.2015.0329. 2016.