Home Page	Contents    Membership    About Cryonics     FAQ     Links     News

Glycerol Perfusion Solution
    for Post-Mortem Cryonics Institute Sign-ups

by Ben Best

Glycerol Perfusion Solution

Glycerol (glycerin) solution can be used for those who wish to have their local funeral director perfuse a cryonics patient that is to be held in dry ice for the two week period required by the Cryonics Institute for post-mortem sign-ups. The perfusion protocol requires 5 liters of 2.0 Molar glycerol, 5 liters of 4.0 Molar glycerol, and 20 liters of 8.0 Molar (8.0 moles per liter) — all in saline (0.91 w/v% NaCl) solution. The sodium chloride will make the perfusion solution isotonic.

Any pharmacy should have glycerol (glycerin) in stock, although perhaps no one pharmacy will have large quanities. For those who do not have the time or expertise to make solutions, the glycerol solutions can be purchased from the Cryonics Institute for $1,000 in North America, or $1,500 elsewhere. The prices include shipping costs. A $200 refund will be given for the return of the empty containers in which the glycerol solutions are shipped. Glycerol solutions will not be shipped until payment has been received by the Cryonicis Institute. Arranging payment and waiting for shipment can cause significant delays that are likely to compromise the condition of the patient by an amount that is hard to quantify. For those who prefer to save time and money by making their own glycerol solutions, the formula for doing so is given below.

The ingredients are very inexpensive and almost all of the cost is for labor, shipping and heavy-duty shipping containers. The patient should be held on water-ice (not dry ice) while waiting for glycerol perfusion. Slow deterioration happens to body tissues even at water-ice temperature. For this reason, as well as to save money, if perfusion is desired it is advisable to determine cost and availability of glycerol and labor for preparing solutions rather than waiting for shipment of glycerol solutions from Michigan. Delays waiting for glycerol perfusion may result in such deterioration damage that it may be preferable to cool immediately to dry ice temperature in order to stop deterioration rather than attempt perfusion.

Glycerol cannot eliminate ice formation, but may reduce it. For storage on dry ice for extended periods glycerol may actually be less damaging than vitrification solution, however. Vitrification solutions are typically "metastable" above the temperature at which they solidify (dry ice temperature, for example). Which means that vitrification solutions can freeze if allowed to sit at those temperatures. Freezing in vitrification solutions at dry ice temperature could be more damaging than freezing in glycerol at dry ice temperature because the glycerol freezing would likely happen outside of cells at higher temperatures, whereas the vitrification solution would more likely result in freezing inside cells at lower temperatures — which is more damaging.

16 avoirdupois ounces = one avoirdupois pound = 454 grams    One avoirdupois ounce = 28.35 grams

REQUIRED:

At least 200 grams of pure sodium chloride (NaCl)
About 16 liters of pure glycerol (glycerin)
About 20 liters of distilled water
3 nine liter (or larger) containers
2 six liter (or larger) containers
Graduated cylinder for careful measurements

STEP 1: Make concentrated salt solution. Add 200 grams of pure sodium chloride (NaCl) to a one liter container and fill the container up to the one-liter mark using distilled water. Do this twice to make two liters of concentrated salt solution. 200 grams of sodium chloride will dissolve completely in one liter of water with patient stirring.

STEP 2: Make 27 liters of 8.0 Molar (8.0 moles per liter) glycerol solution by making 9 liters in three batches. Add 5.26 liters of pure glycerol to a container that can hold 9 liters of solution. Add 410 milliliters (mL) of the concentrated salt solution. Then fill the rest of the volume (to 9 liters) with distilled water — adding approximately 3.33 liters of distilled water. Do this whole procedure three times so that you will have 27 liters of 8.0 Molar glycerol solution.
 
Be careful to pour the glycerol solution slowly and carefully, because pure glycerol is very viscous and air bubbles are slow to rise, and can significantly alter volume measurement. A wider measuring vessel is better than a narrow one. Rinsing with distilled water used for the solution can also help keep volume measurements more accurate. Note that values are approximate because mixing water and glycerol is not as completely additive as adding water to water or glycerol to glycerol. Adding water to get the correct total final volume of 8 liters is the most accurate way of measuring.

STEP 3: Make 9 liters of 4.0 Molar (4.0 moles per liter) glycerol solution. Add 4.5 liters of 8.0 Molar glycerol solution to a container that can hold 9 liters of solution. Add 200 milliliters (mL) of the concentrated salt solution. Then fill the rest of the volume (to 9 liters) with distilled water — adding approximately 4.30 liters of distilled water.

STEP 4: Make 6 liters of 2.0 Molar (2.0 moles per liter) glycerol solution. Add 3 liters of 4.0 Molar glycerol solution to a container that can hold 6 liters of solution. Add 136 milliliters (mL) of the concentrated salt solution. Then fill the rest of the volume (to 6 liters) with distilled water — adding approximately 2.86 liters of distilled water.

RESULT:

22.5 liters of 8.0 Molar glycerol
6 liters of 4.0 Molar glycerol
6 liters of 2.0 Molar glycerol

Only the head is being perfused, because reducing freezing damage in the brain is most important. The glycerol solution is not a vitrification solution — but it will greatly reduce ice formation. For the perfusion, the carotid arteries in the neck are cannulated (cannulae pointing to the head), and the jugular veins in the neck are cut for drainage. Five liters of 2.0 Molar glycerol is perfused first, followed by five liters of 4.0 Molar glycerol, and then twenty liters of 8.0 Molar glycerol. Perfusion pressure should be high enough to see drainage from the jugular vein, but perfusion pressure should not be much higher. Perfusion pressure should be approximately physiological (100 mmHg to 120 mmHg) rather than the higher perfusion pressures often used for embalming.

The molar mass ("molecular weight") of glycerol is 92.09 grams per mole. Thus:
 
92.09 grams glycerol          8 moles glycerol          736.7 grams glycerol
------------------------    X     -------------------    =     ------------------------
      mole glycerol                       liter                          liter solution
 
The density of glycerol is 1.261 grams/cm³ = 1.261 gm/mL. Thus:
 
736.7 grams glycerol            1 mL glycerol             584 mL glycerol
------------------------    X     --------------------    =     ------------------
      liter solution                 1.261 gm glycerol           liter solution
 
Therefore, an 8.0 Molar glycerol solution will contain 736.7 grams of glycerol, which will be 584 milliliters of glycerol per liter of final solution. Nine liters of 8.0 molar glycerol solution will contain
9 X 0.584 liter = 5.256 liters of pure glycerol (5.258 liters if more decimals are used).

Saline solution is 0.91 weight (mass) per volume percent sodium chloride (0.91w/v% NaCl), meaning that there are 9.1 grams of NaCl in one liter of solution. The goal is for the glycerol solutions to contain 9.1 grams of NaCl per liter of solution in order to be isotonic. Nine liters of solution will therefore contain
9 X 9.1 grams = 81.9 grams of NaCl

It is convenient to make a concentrated salt solution for use in making isotonic glycerol solutions. The concentrated salt solution suggested will have 200 grams of NaCl per liter rather than the 9.1 grams of NaCl per liter of isotonic (saline) solution.

81.9 grams NaCl            1 liter conc. NaCl            0.4095 liters conc. NaCl
--------------------    X     ---------------------    =     ---------------------------
  9 liters solution             200 gm NaCl                    9 liters solution
 
Therefore, 410 milliliters (0.410 liters = 410 mL) of concentrated NaCl solution is added to 5.26 liters of pure glycerol when making 9 liters of 8.0 Molar glycerol solution. Adding approximately 3.33 liters of distilled water will give 9 liters of 8.0 Molar glycerol solution because
 
5.26 liters glycerol + 0.410 liters NaCl = 5.67 liters total solution
and
9.00 liters − 5.67 liters = 3.33 liters water (approximately)
 
The values are approximate because mixing water and glycerol is not as completely additive as adding water to water or glycerol to glycerol. Adding water to get the correct total final volume of 8 liters is the most accurate way of measuring.

Diluting 4.5 liters of 8.0 Molar glycerol with an equal portion of water would make 9 liters of 4.0 Molar glycerol solution, but it would not be an isotonic solution. The 9 liters would only contain 40.95 grams (9.1 X 4.5) of NaCl. Another 40.95 grams of NaCl is added by adding about 200 mL of the concentrated NaCl solution.
 
40.95 grams NaCl         1 liter conc. NaCl          0.205 liters conc. NaCl
-------------------     X     -------------------    =     -------------------------
  9 liters solution             200 gm NaCl                9 liters solution
 
Therefore, 200 milliliters (0.200 liters = 200 mL) of concentrated NaCl solution is added to 4.500 liters of 8.0 Molar glycerol when making 9 liters of 4.0 Molar glycerol solution. Adding approximately 4.30 liters of distilled water will give 9 liters of 4.0 Molar glycerol solution because
 
4.50 liters + 0.20 liters = 4.70 liters
and
9.00 liters − 4.70 liters = 4.30 liters water (approximately)

Diluting 3 liters of 4.0 Molar glycerol with an equal portion of water would make 6 liters of 2.0 Molar glycerol solution, but it would not be an isotonic solution. The 6 liters would only contain 27.3 grams of NaCl. Another 27.3 grams (9.1 X 3) of NaCl is added by adding 136 mL of the concentrated NaCl solution.
 
27.3 grams NaCl           1 liter conc NaCl          0.136 liters conc NaCl
--------------------    X     --------------------    =    --------------------------
  6 liters solution            200 gm NaCl                6 liters solution
 
Therefore, 136 milliliters (0.136 liters = 136 mL) of concentrated NaCl solution is added to 3.000 liters of 4.0 Molar glycerol when making 6 liters of 2.0 Molar glycerol solution. Adding approximately 2.86 liters of distilled water will give 6 liters of 2.0 Molar glycerol solution because
 
3.000 liters + 0.136 liters = 3.136 liters
and
6.000 liters − 3.136 liters = 2.864 liters water (approximately)