Cryoprotectants and cell cryopreservation. What are your options?
Once again we find ourselves quoting Henry Ford. He famously once said “Customers can have any color they want as long as it’s black”. Cell biologists have for decades faced a similar alternative: “You can have any cryoprotectants you like as long as it’s DMSO”.
So after all this time we ask the question, is dimethyl sulfoxide (DMSO) still the only practical alternative for cell freezing? Or is there a new kid on the block? We précis the findings of Felipe de Lara Janz et al.1 and discuss the alternative freezing protocols and cryoprotectants available.
Cryoprotectants are an essential ingredient for cell freezing; protecting the cell on its journey from room temperature to -196 °C. The general properties required for cryoprotectants are that they have low molecular weight, are nontoxic and cheap. They also need to provide high levels of cell viability post thaw, and have no other influence on the cells, (i.e., they must not affect the cells in a way that causes them to behave differently after exposure to the cyroprotectant).
Cryoprotectants are divided into two main classes:
- Intracellular agents (e.g., DMSO, Glycerol, PEG), which penetrate inside the cell preventing the formation of ice crystals that could result in membrane rupture.
- Extracellular agents that do not penetrate in cell membrane and act to improve the osmotic imbalance that occurs during freezing (e.g., sucrose, trehalose, and dextrose)
So which is better? Intracellular or extracellular? The paper by Felipe de Lara Janz et al., does a good job of separating out the effects of the different cryoprotectants experimentally and arrives at the conclusion that intracellular agents are required for optimal cryopreservation1.
“Cells preserved in sucrose and trehalose alone had a low rate of stem cell viability and, consequently, an increase in lag phase after thawing, maybe because these extracellular agents do not prevent the formation of ice crystals within the cell” states Felipe de Lara Janz et al“1
But while we need the intracellular agents in order to adequately preserve the cells, there are well documented downsides to DMSO and the other intracellular agents. Several studies have indicated that DMSO can induce neuronal differentiation in stem cells.2 This coupled with the cellular toxicity of DMSO at room temperature has lead to the search for a new cryopreservant that provides the same level of benefit as DMSO but without the drawbacks.
Other authors have discussed the potential for DMSO replacements, or at least compounds that can be used alongside DMSO to reduce its use in the critical environment of stem cell freezing. Hunt et al., discusses the use of alternatives including polyvinylpyrrolidone (PVP), methylcellulose, and others either alone or in the presence of reduced levels of DMSO.3 The authors conclude that there is “potential for the development of xeno-free cryoprotectant solutions utilizing lowered concentrations of DMSO and slow cooling.”3
It summation while there are alternative cryoprotectants, none are truly capable of replacing DMSO as the gold standard. Whilst DMSO does have some well characterized drawbacks there is no direct replacement for it in cell freezing. For the moment at least it truly is one choice. I hope you like black!
References for Cryoprotectants
- Evaluation of Distinct Freezing Methods and Cryoprotectants for Human Amniotic Fluid Stem Cells Cryopreservation. Felipe de Lara Janz,1 Adriana de Aguiar Debes,2 Rita de Cássia Cavaglieri,1 Sérgio Aloísio Duarte,3 Carolina Martinez Romão,1 Antonio Fernandes Morón,4 Marcelo Zugaib,3 and Sérgio Paulo Bydlowski. Journal of Biomedicine and Biotechnology. Volume 2012 (2012), Article ID 649353, 10 pages. doi:10.1155/2012/649353
- K. Mareschi, D. Rustichelli, M. Novara et al., “Neural differentiation of human mesenchymal stem cells: evidence for expression of neural markers and eag K+ channel types,” Experimental Hematology, vol. 34, no. 11, pp. 1563–1572, 2006.
- Cryopreservation of Human Stem Cells for Clinical Application: A Review. Charles J. Hunt.. Transfus Med Hemother. 2011 April; 38(2): 107–123. Published online 2011 March 16. doi: 10.1159/000326623. PMCID: PMC3088734