Oregon Cryonics
A Non-profit Organization

Manual - Cryoprotection Protocol

Back to Manual - Perfusion

 

Solution Mixing

Mix and store all four solutions ahead of time according to the worksheets.  Store the worksheets with the containers of solution in refrigerator.  Shelf life is about one year.  They only need to be refrigerated so that the solution will be cold when needed, but not to maintain quality.

 

Washout

Water, PBS, sodium azide 0.1%, heparin.

This is an isotonic buffered solution for washing out the blood.  The sodium azide immediately halts mitochondrial respiration and the associated cascade of damage that would otherwise follow.

Perfuse at 100 mmHg until effluent is sufficiently clear.  Only part of the volume will be used.

 

Fixative

NBF 10%. Glutaraldehyde (GA) 3% is added at time of use.

Switch valves to the fixative container. 

Perfuse 4 L over approximately 30-45 minutes at very low pressure.

 

Carrier

Water, PBS, sodium azide 0.02%, Sodium Dodecyl Sulfate(SDS) 0.01%. GA 3% is added at time of use.

Formalin is not present in the carrier because it is not compatible with the DMSO in the CPA. 

The SDS opens the BBB to allow CPA to move into the tissue without causing dehydration and mechanical distortion.

Switch valves to the carrier container. 

Perfuse at the slow rate of about 2 L per 15 minutes. So only about 3 L will be used because of the ramp.

 

Cryoprotectant (CPA)

Water, PBS, DMSO 37% v/v, Ethylene Glycol 37% v/v. Total 74% v/v, or 82% w/v.

GA 3% is added at time of use.

Start ramping in the CPA with the second pump to reach 100% at about 30 minutes.

Continue at 100% at 2L per 15 minutes for another 30-60 minutes.