Biobanking Science: New Non-Toxic Cryoprotectant Solutions

Stem cells cryopreserved and biobanked in new osmolyte solutions show better post-thaw structure and function than cells frozen in DMSO.

Biobanking can cause significant freezing and thawing stress to cells.
Cryoprotectants help cells cope with freezing and thawing stress during biobanking.
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Cell therapy is a rapidly growing field that uses live cells to treat patients. This presents unique biobanking challenges because, unlike traditional pharmaceutical products, cells must be alive and functional to be effective therapeutics. Unless cell therapy cells are harvested and immediately administered to patients, they must be frozen and stored in biobanks under strict temperature-controlled conditions to maintain cellular viability and function.

Cell therapies based on mesenchymal stromal cells (MSCs) are currently stored in many biobanks around the world and are being investigated in hundreds of clinical trials. MSCs can differentiate into many different cells including cartilage, bone and fat cells. They can also modulate the patient’s natural immune system. Therefore, MSC-based cell therapies may be able to repair damaged tissues and treat immune disorders like Crohn’s disease. MSCs are an attractive cell therapy option because they are easy to harvest from adult bone marrow and adipose tissue. In most cases, MSCs are collected and then cryopreserved in biobanks for future research or patient administration.

Biobanks Need New Cryoprotectant Solutions

Cryoprotectant solutions must be added to biobanked cells before cryopreservation to protect against the stress of freezing and thawing. DMSO is the most commonly used cryoprotectant in biobanks as it lowers the freezing temperature of water and helps prevent cellular damage from ice crystal formation. However, DMSO is toxic to cells and to patients. Cryoprotectant solutions containing DMSO can impair cell structure and function by degrading macromolecules, altering cellular metabolism, causing epigenetic changes and disrupting the actin cytoskeleton. DMSO can also cause systemic toxicity if administered to patients and may lead to serious side effects such as cardiac arrest and renal failure. These negative effects of DMSO are believed to have caused at least one Phase III clinical trial failure of an MSC-based cell therapy.

Nature Can Help Biobanking Scientists

Researchers from the University of Minnesota are developing a new class of non-toxic cryoprotectant solutions called multi-component osmolyte solutions. These solutions contain sugars, sugar alcohols and small molecule additives, and are based on molecules called osmolytes that are upregulated in natural organisms during environmental stress.

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In a recent publication, researchers compared MSCs frozen in 10% DMSO to fresh cells and to cells frozen in different osmolyte solutions. They tested osmolyte solutions containing sucrose, glycerol and creatine (SGC); sucrose, glycerol and isoleucine (SGI) or sucrose, mannitol and creatine (SMC). MSCs were incubated in these solutions for 1 to 2 hours. Cells were then cooled to -100°C using a controlled-rate freezer and transferred to a liquid nitrogen vapor-phase freezer for biobanking.

Using this protocol, the researchers found that cells biobanked in osmolyte solutions had better post-thaw structure and function than cells frozen in DMSO.

Freezing Rates Matter in Biobanks

Many studies have shown that the rate at which cells are cooled affects ice crystal formation inside and outside the cells. Ice crystals can damage cells and affect post-thaw cellular viability and function, two factors crucial to manufacturing effective cell therapies. The biobanking industry standard is to use controlled-rate freezing to cool cells at a rate of -1°C/minute. This rate has been shown to result in better post-thaw recovery of cells.

However, in this study, researchers used a controlled-rate freezing protocol that included a rapid cooling and warming step to promote extracellular rather than intracellular ice formation. They found that this method resulted in less variability in post-thaw cellular function. They also found that cells cooled in osmolyte solutions at a rate of -3°C/minute had better post-thaw function than cells frozen at the biobanking industry standard of -1°C/minute. These results indicate that more research needs to be done to discover the optimum freezing rate for different cell types.

All cells in this study were rapidly thawed in a 37°C water bath.

Osmolyte Solutions are Less Damaging Than DMSO

With any new cryoprotectant solution, there is always concern that it could damage the cells, alter gene and protein expression or cause them to function differently. The authors tested all these concerns with their new osmolyte solutions. They found that osmolyte solutions do not alter cell surface markers, cell attachment, proliferation, differentiation or cell size of MSCs. In contrast, cells frozen in DMSO were significantly smaller, and showed disrupted actin cytoskeleton and epigenetic changes compared with fresh cells.

MSCs retained their mesenchymal lineage, and their ability to differentiate into different cell types, after being preserved in osmolyte solutions. This is a very important result because cells must maintain their original functional capacity to be effective cell therapeutics. MSCs biobanked in osmolyte solutions also had better post-thaw attachment and actin skeleton alignment than cells frozen in DMSO. They showed less epigenetic changes than cells biobanked in DMSO solutions.

The researchers also looked at gene expression using RNA sequencing and real-time RT PCR. Cells in osmolyte solutions had higher expression of cytoprotective genes such as anti-apoptotic genes and cell adhesion molecules, and lower expression of stress genes than cells biobanked in DMSO.

Conclusion

This study shows that multi-component osmolyte solutions may be less toxic cryoprotectants than DMSO solutions. With further research, biobanks may be able to use these solutions to improve post-thaw function of cells, which is a critical part of developing successful cell therapeutics.

However, this study was done on a cultured MSC cell line. Before biobanks can use osmolyte solutions, further studies are needed to test these solutions with patient-derived MSCs and with other cell types.

 

Reference

Pollock et al. Improved Post-Thaw Function and Epigenetic Changes in Mesenchymal Stromal Cells Cryopreserved Using Multicomponent Osmolyte Solutions. Stem Cells Dev. 2017