Higher concentration of cryoprotectant

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jordansparks
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Higher concentration of cryoprotectant

Postby jordansparks » Tue Jan 23, 2018 9:41 am

I've decided it's time to increase the concentration of cryoprotectant that we use. We've been using 65%, but we're going to move closer to 75% or 80%. Higher concentrations have the very important advantage completely preventing all ice formation, regardless of temperature or cooling rate. This will also allow safe storage at intermediate temperatures of about -125 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.

jordansparks
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Re: Higher concentration of cryoprotectant

Postby jordansparks » Fri Apr 06, 2018 3:01 pm

The simple experiments were done and the results are fascinating.
http://www.oregoncryo.com/researchHighConcentrations.html
Ethylene glycol, as used in Aledhyde-Stabilized Cryopreservation (ASC), is a poor choice of cryoprotectant because of its narrow safe concentration zone. Our EG/DMSO mixture is more similar to Alcor's M22 and CI's VM1, and it's a better choice after aldehyde stabilization. Based on the results, 75% was chosen as a safe concentration for our protocol, with lots of margin for error.

DetnPx
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Re: Higher concentration of cryoprotectant

Postby DetnPx » Sun Apr 08, 2018 12:50 pm

50/50 Ethylene glycol / DMSO:
5% increments, cooled to -80°C. Concentrations of 55% and below were frozen, while 100% was partly frozen. Ranges from 60% to 95% remained clear and syrupy, not very viscous at all. -80°C is just not cold enough.


Did you, coincidentially, also leave these vials at -80C for a prolonged period of time, maybe even up till today? Since this is somewhat similar to international shipments to CSP's, which can sometimes have a delay of 4-14 days before final cooldown to Tg. Prolonged exposure to a temperature above Tg may still inhibit ice crystal growth. I'm hoping you still have the samples at -80C to be able to make a judgement.

jordansparks
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Re: Higher concentration of cryoprotectant

Postby jordansparks » Sun Apr 08, 2018 1:36 pm

We left the vials at -80C for days, without any change. Any crystals formed either immediately or within about a day. I was really struck by how syrupy the mixtures remained. Shipping at that temperature would be a liquid state, not a solid state.

DetnPx
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Re: Higher concentration of cryoprotectant

Postby DetnPx » Sun Apr 08, 2018 1:51 pm

I concur, this could be another serious concern that needs some added research. Thank you for raising the issue.

jordansparks
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Re: Higher concentration of cryoprotectant

Postby jordansparks » Sun Apr 08, 2018 1:59 pm

On the other hand, if no cryoprotectant was used, as in the case of a straight freeze, then we really are talking about an essentially solid state. In a straight freeze situation, -80C is fine for days/weeks.
You know, if a quality fixation was performed on your end prior to cryoprotection, then the liquid state would be much less of an issue. The chemical crosslinking could stabilize well enough for transport. I really don't think there can be two separate perfusions. All perfusion of fixative and CPA would still need to be performed prior to shipment. That's a big technical demand, but it is at least achievable.

jordansparks
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Re: Higher concentration of cryoprotectant

Postby jordansparks » Mon Apr 09, 2018 2:04 pm

It was pointed out that I should have expressed everything as w/v instead of v/v. Yes, of course. So I just went back and revised the website to reflect w/v. And our chosen percentage is now 82% w/v. There might also be room for further improvement. For example, a higher percentage of DMSO and a lower percentage of EG might have some advantage in being less viscous near 0degC as well as being more penetrating.


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