Cryopreservation and Thawing of Human MSCs


A scientist carries out stem cell research in an FDA laboratory at the National Institute of Health in Bethesda, MD. Image credit:

A comprehensive new protocol published out of the Cardiology Stem Cell Centre in Copenhagen, Denmark[1] cites MedCision’s ThawSTAR® cell thawing system as an integral component of their new methodology.

Human mesenchymal stromal cells (hMSCs), as most of our readers know, form the basis of a number of exciting new cell-based therapies currently under clinical investigation. Their therapeutic potential is grounded in their immunosuppressive and regenerative properties. Since nearly all scientists agree that cryopreservation is a practical necessity for “on demand” cell therapy, contemporary cryopreservation and thawing methods must be geared towards protecting these healing properties.

The researchers in Copenhagen define a successful clinical stem cell cryopreservation protocol as one that takes several different factors into consideration, namely cryopreservation media, cryoprotectant agents (CPAs), the freezing container, the freezing temperature, and the cooling and warming rate.

When choosing a suitable cryoprotectant, one of the toughest challenges is finding one that is able to penetrate cells, yet still has low toxicity. DMSO is, of course, the most widely used cryoprotectant, due to its high membrane permeability and impressive track record in protecting cell viability. However, DMSO is toxic at high levels, and therefore its use in a clinical setting necessitates post-thaw washing or dilution steps before cells can be administered. For this reason, alternative cryoprotectants are sometimes used either in place of DMSO, or, more commonly, in combination with it in order to lower the initial DMSO concentration.

Cooling rate is also a significant factor in hMSC viability; the cooling rate must be carefully controlled to avoid the ravages of intracellular ice formation. The authors recognize that passive freezing is more economical than using controlled rate freezers, and specifically recommend an isopropanol free passive freezing container.

Since the temperature at which frozen cells are stored affects their viability, the authors recommend transfer to liquid nitrogen within a day of freezing, and storing at -196 °C, a temperature at which chemically driven degradation is not known to occur.

Thawing rate is just as important as freezing rate in protecting cell viability. The authors outline the use of a traditional warm water bath for thawing, but are careful to note that technology that controls the warming rate more reliably is also available; specifically MedCision’s ThawSTAR system. ThawSTAR is now available in a wider range of vials formats, increasing the system’s suitability for clinical use.

The authors finally caution that initial post-thaw viability numbers don’t always reflect true cell recovery and function. “Invisible” damage can lead to apoptosis and cell death, which is why it’s so important to protect cells from unforeseen temperature fluctuations. While early clinical studies sparked some debate over whether cryopreserved MSCs are the functional equals of freshly isolated cells, more recent results are helping to put these fears to rest. [2] It seems logical that rather than expressing apprehension that cryopreserved MSCs may not retain the functional capacity of freshly isolated cells, we should be advocating to incorporate technology that ensures cryopreserved is as good as fresh—and in fact better, since that functional capacity is preserved for future applications.

[1] Haack-Sorensen M., et al. Cryopreservation and Revival of Human Mesenchymal Stromal Cells. Mesenchymal Stem Cells: Methods in Molecular Biology 1416: 357-374 29 May 2016.
[2] Gramlich, O. W., et al. Cryopreserved Mesenchymal Stromal Cells Maintain Potency in a Retinal Ischemia/Reperfusion Injury Model: Toward an off-the-shelf Therapy. Nature: Scientific Reports 6, Article number: 26463, 1-12. 2016.

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