I’ve queries regarding cryopreservation of mammalian cells in DMSO the queries are as fallows:
Q) How DMSO will protect cells from intracellular ice formation during freezing?
What I know is It will penetrate into the cells the push out the water from cells so that no ice crystals are formed , If this is the case then
Q) During freezing DMSO will penetrate inside the cells and displace the water out from the cells thereby preventing ice crystal formation inside the cells, But the water that was displaced out from the cell will form ice crystals during freezing, So won’t they cause any damage to cells (i.e damage caused due to ice formation by extracellular water)?
Q) DMSO is toxic to cells, But during freezing the DMSO will enter inside the cells in that case won’t it be toxic to cells (I mean DMSO outside is toxic but inside is not toxic to cells)?
Q) During freezing DMSO will penetrate inside the cells and displace the water out from the cells, Whereas during Thawing water penetrate inside and DMSO comes out of the cells why (or) what is the mechanism?
Q) During freezing is there any residual DMSO that was left outside the cell (or) entire DMSO will penetrate into the cells? (I mean During cryo preservation will the entire DMSO penetrate into the cells (or) only some portion will enter and some other portion will remain outside the cell)?
Q) Why DMSO enters into cell during freezing and why it comes out of the cell during thawing?
Q) During Thawing why the penetrated DMSO into the cells will come out? Why can’t it remain inside the cell?
Q) During thawing is there any residual DMSO that was left inside the cell?
Q) During cryopreservation at low temp all the metabolic activities get’s stopped, Then why we have to include Complete Media/ 95% FBS along with cryoprotectant (Such as DMSO), Why can’t we simply cryo preserve in DMSO?
Thank You.
Hello Vikram Kumar,
Q1) DMSO when added to the cell media, commonly at 2 M concentration, increases the porosity of the cellular membrane, which allows water to flow more freely through the membrane. Additionally, like glycerol, DMSO is thought to help prevent the formation of water crystals by increasing intracellular solute concentration, thus aiding in the prevention of intracellular ice formation (PMID: 33757335)
Q2) While extracellular water displaced by DMSO can form ice crystals during freezing, the presence of DMSO reduces the formation of large, damaging ice crystals. The slow freezing process (-1ºC per minute) also helps prevent the formation of intracellular ice crystals by allowing sufficient efflux of water before freezing. (PMID: 33757335, 18080461)
Q3) DMSO can be toxic to cells at higher concentrations or prolonged exposure. However, during cryopreservation, lower concentrations and limited exposure mitigate its toxicity. (PMID: 34630858, 24327606)
Q4&6&7&8) During freezing, DMSO enters cells, displacing water. During thawing, DMSO diffuses out while water enters the cells, driven by concentration gradients and changes in cell membrane properties. There may be some residual DMSO left inside the cell after thawing, but it is diluted and removed from the immediate environment of the cells due to the hypertonic environment created by the water entering the cells. (PMID: 33757335, https://handling-solutions.eppendorf.com/cell-handling/faqs/how-does-dmso-affect-your-cells/)
Q5) Not all the DMSO penetrates into the cells during cryopreservation. Some DMSO remains outside the cells, and the amount can vary depending on factors such as cell type and cryopreservation protocol. (https://handling-solutions.eppendorf.com/cell-handling/faqs/how-does-dmso-affect-your-cells/ ; https://www.abcam.com/protocols/cryopreservation-of-mammalian-cell-lines-video-protocol)
Q9) Including a complete medium or serum components like FBS along with DMSO serves purposes such as cell survival, recovery, and subsequent growth after thawing. (PMID: 19788372, 31279341)
Sincerely,
YS
Here is a nice review:
Article Dimethyl sulfoxide: a central player since the dawn of cryob...
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