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We use 82% w/v cryoprotectant concentration.  This is considered to be a very high concentration. It has the very important advantage completely preventing all ice formation, regardless of temperature or cooling rate. This also allows safe storage at intermediate temperatures of about -130 °C without any risk of ice nucleation. This approach is known as equilibrium vitrification, and it only works for us because we chemically fix the tissue first, so toxicity at the higher concentration is not an issue.

 

We took some time to document the range of concentrations that tend not to freeze for various cryoprotectant mixtures.  We use this information to help select concentrations that tend to prevent ice formation during cases.  The concentration can't be too high, because then the other components of the cryoprotectant can freeze.

 

Methods

A series of vials were filled to 6 mL with mixtures of various concentrations of cryoprotectant in water.  5% or 2% increments were prepared, ranging from about 45% to 100% w/v.  Cooled to either -80°C on dry ice or to -135°C in an LN2 freezer.

 

Results and Discussion

Glycerol:

6% increments, cooled to -80°C.  Crystals were observed in concentrations of 82% w/v and lower.  Concentrations between 88% and 100% w/v remained clear, and turned hard and gummy.  We know that the more concentrated mixtures of glycerol could grow crystals if seeded, but we didn't do so because we don't use glycerol.

 

Ethylene glycol:

5% increments, cooled to -80°C.  Chalky white crystals were clearly observed in concentrations of 67% w/v and lower, as well as 89% w/v and higher.  In most cases, the crystals very obviously turned the entire vial solid white.  While we did not bother to narrow the range any further, the observed safe range with no crystals was between 72% and 83% w/v.  This was a surprisingly narrow range.  Also, since Aldehyde-Stabilized Cryoprotection (ASC) is being advodated at 65% w/v, we observe that ice crystals should be forming at that concentration.  Even at higher concentration, this does not seem like a particularly useful cryoprotectant because the range is too narrow.  It's very easy to envision areas that have slightly inadequate perfusion forming ice crystals.

 

50/50 Ethylene glycol / DMSO:

5% increments, cooled to -80°C. Concentrations of 61% w/v and below were frozen.  66% w/v and above remained clear and syrupy, not very viscous at all. -80°C is just not cold enough.

 

50/50 EG/DMSO, 3% glutaraldehyde, and phosphate buffer:

2% increments from 58% to 93% w/v.  We did not go above 93% because the reduced water made it impractical to mix in the glutaraldehyde and buffer.  Cooled to -135°C. Concentrations of 60% w/v and below had ice crystals.  62% through 93% w/v were clear and solid.  This mixture is what we use for patients.  Our patient concentration of 82% w/v is well inside the safe range, being 18% higher than needed.  This provides a margin of error for areas that might be poorly perfused and so might be at a lower concentration.