Cryopreservation of mesenchymal stromal cells can attenuate clinical immune effects

by Alexey Bersenev on May 10, 2014 · 7 comments

in cell product,mesenchymal

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As Jacques Galipeau reported in conferences and in the paper, cryopreservation could negatively affect therapeutic “immunomodulatory value” of mesenchymal stromal cells (MSC). There was no independent confirmation of Galipeau’s findings, and many MSC product developers remained skeptical. This week, Katarina Le Blanc published a report, which supports Galipeau’s conclusions and provides more insight into potential clinical value of this phenomenon. Let me just say – this paper could change the field!

Le Blanc concluded that freeze-thawed human MSC compared to fresh MSC possess:

  1. increased triggering of Instant Blood Mediated Inflammatory Reaction (IBMIR);
  2. strong activation of complement and coagulation cascades;
  3. increased sensitivity (lysis) to exposure of normal human serum;
  4. overall reduced immunomodulatory and blood regulatory properties;
  5. trend toward worse clinical response in GVHD clinical trials.

I’d like to summarize more clinical part of the study. 44 infusions of MSC were analyzed retrospectively. 9 of 44 were fresh MSC infusion, the rest freeze-thawed. Conclusions from clinical part:

  • patients with fresh + early passage (1-2) MSC infusions showed 100% response rate
  • patients with freeze-thawed + later passage (3-4) MSC infusions showed 50% response
  • however this difference was not statistically significant (p=0.06)
  • viability was the same between fresh/ cryopreserved MSC and between responders/ non-responders
  • there was no difference in long-term engraftment of MSCs in tissues between fresh/ cryopreserved groups.

Significant limitation of clinical part of the study, mentioned by authors in discussion, is low number of comparable infusions (fresh n=9). This limitation affected statistical power of the study. The authors wrote:

A power analysis aiming at 80% power at the 0.05 significance level shows that we can only expect statistical significance for difference n clinical effect of more than 50%, meaning that we might ignore clinically relevant differences. We must therefore also consider trends, even though this could be considered speculative.

The authors performed a number of in vitro studies, testing immunomodulatory and blood regulatory properties of fresh versus frozen MSCs. Some interesting findings from this part:

  • strong compliment lysis by normal human serum of cryopreserved MSCs was abrogated by addition of EDTA;
  • despite the fact that level of IDO upon interferon-gamma licensing was much higher in fresh MSC, there was no difference in other immunomodulatory cytokine – IL-6;
  • even though frozen MSCs were inferior to fresh in PHA-stimulated MLR, there was no difference in allo-stimulated MLR.

Overall, I think, this study is invaluable! Everyone, who is involved in MSC-based clinical trial must read it! Le Blank study confirms earlier finding by Galipeau’s group and could have a big impact on a field. As of now, we don’t have convincing positive results from Phases 2/3 of clinical trials, where MSCs are used as powerful immunomodulators (GVHD, Crohn’s disease). Contrary, we have a history of big failures of MSCs in “immunomodulatory trials”. For example, Osiris Phase 3 GVHD trial and very recent Athersys Phase 2 IBD trial. All clinical trials right now are utilizing frozen MSCs! Could a phenomenon, describing by Galipeau and Le Blanc, be a real deal breaker? Very much possible! I guess we will know for sure with more independent studies and with direct comparison of fresh versus frozen in clinical trials. Galipeu just started a trial for Crohn’s disease with freshly harvested MSCs. I hope they can include a small “frozen MSC” group.

{ 7 comments… read them below or add one }

Augustine May 12, 2014 at 1:16 am

it may be possible to use fresh MSC for infusion to elective patients but for GvHD patients it may not be possible unless there is a harvest at the time of onset.

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Alexey Bersenev May 12, 2014 at 10:41 pm

I think, fresh is logistically difficult, but entirely possible for almost any GVHD. First, for treatment of chronic steroid-resistant GVHD and for prophylaxis – you have enough time to find a donor and generate a product. Second, even for acute GVHD you can have 2-3 weeks to make a product in case if you don’t need HLA match or if you have a large HLA database. Make a products means thaw banked marrow from healthy donor and culture MSC –> infuse fresh.

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Vlad May 12, 2014 at 5:25 am

Hi Alex. I know article where authors did not detect any difference between frozen/fresh MSC or 1/2 passage MSC vs 3-4. I think problem is more complex and numerous studies are needed. http://www.ncbi.nlm.nih.gov/pubmed/23997985

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Alexey Bersenev May 12, 2014 at 11:05 pm

Thank you for bringing this up, Vlad. I’ve missed this study. I don’t think these 2 studies contradict each other, since Le Blanc study did not compared frozen versus fresh (without adding passages number) directly. If they did, it will be far from significant (= no difference) (correction 05/13/14: they compared all passages versus 1-2 for fresh and frozen), since even frozen/ passage 3-4 versus fresh/ passage 1-2 difference is not significant.

The passage number impact will contradict to conclusions from earlier Le Blanc study, and this is interesting twist.

I’m completely agree with you, that problem is more complex than we think. There should be more independent studies to make solid conclusions. A lot of factors can play potential role in different results between groups, for example:
- the method of cryopreservation (cryo- solution, processing and storage before…)
- the method of thawing before infusion
- cell culture protocol
- patients heterogeneity and enrollment criteria (as Resnick said in the paper – response rate, cited in literature from 0% to 100%)
and bunch of other factors.

But what the most important in Galipeau and Le Blanc studies is bringing attention of community to this issue. Why I think it could change the filed? Because (1) every developer now have get potency assay ready even in pre-clinical stage and (2) every developer now must test and find the best cryopreservation protocol or decide against it.

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Vlad May 15, 2014 at 3:12 am

Alex. Do you now any good potency assay for MSC immunomodulation? IDO, TSG6 and other mRNA expression? PHA-stimulated MLR/allo-stimulated MLR? Do these assays correlate with clinical outcomes??

Most intriguing for me in this article – high level dead/apoptotic MSC (fresh and frozen) in normal human serum (more than 50%-70??). Why is this happening?
What should we do to prevent this after i/v MSC infusion?

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Chris Centeno May 12, 2014 at 3:33 pm

Excellent post Alexey. As you know, I’ve also had a concern that many of these commercial ventures are growing cells out far beyond what any existing clinical study would support in the name of reducing manufacturing costs.

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Pedro Silva Couto June 20, 2014 at 7:35 am

Dear Dr. Alex,

That was a nice review from you regarding this important papper! I was reading carefully the article and the coments posted here and I my opinion is:

-Thinking in terms of logic, fresh MSC might be better than frozen ones (less manipulation might be seen as better quality).

-Objectively: They used 10% of DMSO as cryoprotectant agent (CPA)! This is a too simple approach once in stem cell banking CPAs used are usually complexed mixtures of DMSO and Dextran among others. Moreover It is known that CPAs used as well as freezing protocols (for instance slow VS fast cooling protocols) affect cell viability and potency! We can speculate wether they influence also the immunomodulatory properties of MSC.

-When they compared the therapeutic efficacy it seems unfair to me: they compare fresh MSC in P1-P2 with freeze-thawed cells in the passage P3-P4. It is known that with prolonged culture periods the cells became older and start to age…so I believe that this comparison it is not completely conclusive.

Overall I believe that more studies are required to extract a final conclusion regarding fresh VS frozen MSC!

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