DMSO-Free Cryopreservation of umbilical cord MSCs – New Study, Old solutions: Do they work the same?

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Many studies have addressed the deficiencies of DMSO as a cryoprotectant and addressed this by developing cocktails of solutions that replace DMSO with safer alternatives that provide both cryoprotection and high functionality of the stem cells.

Mesenchymal stem cells (MSCs) have enjoyed a particularly high attention in the cryopreservation literature, as many studies have reported about new approaches to cryopreserve these cells without DMSO.

Most of these studies have used mesenchymal stem cells derived from bone marrow or adipose tissue.

A new report by Dr. Gyu-Jin Rho’s lab at Gyeongsang National University in Korea published in the International Journal of Stem Cells recently addressed these previous studies by applying a group of commonly used non-DMSO cryoprotectants to the cryopreservation of mesenchymal stem cells from Human Wharton’s Jelly (WJ). These are essentially umbilical cord-derived cells that have been demonstrated as potentially valuable in the treatment of many diseases.

The study replaced DMSO with mixtures of polyvinylpyrrolidone, ethylene glycol, glucose, sucrose and  FBS. Additionally, the authors tried to develop these same mixtures without FBS.

For cryopreservation, the cells were equilibrated for 30 min at 1°C, then cooled according to the following protocol: −2°C/min to −9°C, then −9°C to −9.1°C and held for 5 min; then −0.3°C/min to −40°C; then −10°C/min to −140°C before being placed in liquid nitrogen for storage up to 3 months.

Readouts included viability, adipogenic and osteogenic differentiation as well as expression of transcriptional factors, apoptosis-related genes and lineage-specific markers.

Below are the solutions used.

CPA Solution 1: 10% (v/v) PVP in ADMEM with 10% (v/v) FBS

CPA Solution 2: 10% (v/v) FBS in ADMEM with 0.05 M glucose, 0.05 M sucrose and 1.5 M ethylene glycol

CPA Solution 3: 10% (v/v) DMSO in ADMEM with 10% (v/v) FBS

CPA Solutions 4/5/6: Similar to Solutions 1, 2 and 3 respectively but without 10% (v/v) FBS

So what about the results? In short, DMSO performed the best, but not by much. Viability obtained with the DMSO-containing was 81.2%, around 75% for Solution 2 and 68.2% for solution 1. In all cases, these numbers dropped significantly when FBS was removed from the solutions.

Elsewhere, there was no significant difference found in the expression of cell surface antigens between cryopreserved and control groups.

These results suggest that DMSO-free alternatives that include components that support cell viability and reduce apoptosis are valuable options for the preservation of WJ-derived MSCs. The authors postulate that completely xeno-free solutions will need to include alternative protein sources that can fulfill the role that FBS plays.

The study can be accessed here.

And if you are in the market for DMSO-free options for your applications or just need more information about what the best solution for your situation is, please contact us so we can lend our extensive experience in DMSO-free cryopreservation to helping you.

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