Message: #2643 - About BPI Tech Brief 5
Date: 12 Mar 94 20:33:51 EST
From: Mike Darwin <>
Message-Subject: SCI.CRYONICS About BPI Tech Brief 5


Posted elsewhere on Cryonet and SCI.CRYONICS is BPI Tech Brief #5.  This
paper is self explanatory.  Unfortunately a limitation we still face with
current E-mail technology is the inability to show illustrations with
text.  This is especially unfortunate in a situation such as this one
where understanding just what is going on is more than a little dependant
on being able to see pictures or drawings.  It is our hope that a viable
technical journal dealing with the topics of suspended animation, CNS and
human cryopreservation will soon emerge and that papers such as this one
will be found worthwhile to be published there.  In the meantime, please
bear with us.



by Michael G. Darwin and Steven B. Harris, M.D.


      Permeable cryoprotectives used to achieve colligative 
cryoprotection of human cryopreservation patients are 
osmotically active.  In order to minimize injury from the 
perfusion of these agents in hyperosmolar concentrations it 
is necessary to gradually increase their concentration 
during the course of cryoprotective perfusion (1).  This 
allows time for the agent(s) to diffuse across the capillary 
and cell membranes and gradually exchange with both 
intracellular and interstitial water. Introduction of 
cryoprotectant(s) at too rapid a rate causes cellular 
dehydration resulting in direct injury to the cell membrane 
as well as disruption of cell-cell connections. 

      Achieving a slow, controlled rate of cryoprotectant 
introduction is most commonly achieved by the linear gradual 
addition of perfusate containing concentrated 
cryoprotectant(s) to a recirculating system (which 
initially) contains perfusate which is isosmotic with, and 
circulated through the vascular system of, the patient or 
organ to be cryoprotected (2,3).

      A schematic of such a system which has been employed 
by the Alcor Foundation to achieve cryoprotective perfusion 
is shown in Figure 1.  The system consists of two reservoirs 
and two pumps:  One reservoir, the concentrate reservoir, 
contains perfusate which consists of salts, sugars, colloid 
and buffer dissolved in a concentrated solution of 
cryoprotectant and water.  Commonly this would be a solution 
made up of perfusate components as shown in Table 1 
dissolved in a 65% (v/v) solution of glycerol in water.  The 
second, or recirculating reservoir, contains the base 
perfusate with either little or no added cryoprotectant.  
The contents of the concentrate reservoir are then added 
gradually to the recirculating reservoir while, at the same 
time, an identical amount of perfusate is withdrawn and 
discarded from the recirculating system.  

      One pump, the arterial pump, is used to pump perfusate 
from the recirculating reservoir through the patient from 
where it returns to the recirculating reservoir by gravity 
drainage.  The second pump serves to add perfusate 
containing concentrated cryoprotectant to the recirculating 
system (from the concentrate reservoir) while at the same 
time withdrawing an identical amount of the recirculating 
perfusate (usually drawn from the more dilute venous return) 
and discarding it.  In this way the concentration of 
cryoprotectant is gradually increased in the recirculating 
system (including the patient) until the desired terminal 
concentration of agent is achieved, usually 4M to 6M 
glycerol.  A detailed mathematical analysis and computer 
model of this system of cryoprotectant introduction has been 
done by Perry (4).

*Illustration Not Shown.

FIGURE 1: Typical Cryoprotective Perfusion Circuit

1) Concentrate Reservoir           
 2) Recirculating Reservoir
9) Stopcock     
10) Arterial Cannula

3) Arterial Pump                        
 4) Pulsatile Flow Generator
11) Venous Cannula  
12) Venous sample line

5) Oxygenator 
6) Heat Exchanger
13) CPA add/withdrawl pump 
14) Drain

7) Arterial Filter  
8) Sample manifold
15) Cardiotomy sucrtion  
16) Cardiotomy pump

17) Vent line to recirc. 

18) Magnetic stirrer



Component               Molar Concentration                                                             



Mannitol                         170.00  (182.2)

Adenine HCl                      0.94 (MW 180.6)                  

D-Ribose                           0.94 (MW 150.2)                 

Sodium Bicarbonate          10.00 (MW 84.0)        

Potassium Chloride           28.3  (MW 74.56)       

Calcium Chloride                1.0  (MW 111)        

Magnesium Chloride           1.0  (MW 95.2)                  

Sodium HEPES                30.0  (MW 260.3)              

Glutathione (free acid)        3 .0 (MW 307.3)          

Hydroxyethyl Starch                   ----                    
50.00 g/l   

Glucose                             5.0 (MW 180.2)  

Heparin                                      ----                   
1,000 IU/l         



      The introduction of concentrated cryoprotectant 
solution into the recirculating perfusate in such a way as 
to minimize osmotic stress requires that the concentrate be 
rapidly and completely mixed with the perfusate present in 
the recirculating reservoir.  This is especially important 
since the concentrate has a higher specific gravity than the 
recirculating perfusate. A consequence of this is that the 
added concentrate solution will sink to the bottom of the 
recirculating reservoir where, depending upon the mechanics 
of the system, it will either remain as a static, unmixed 
layer or, if the intake line of the arterial pump originates 
at the bottom of the recirculating reservoir, (a desireable 
place for it since this minimizes the chance of pumping air 
to the patient) serve as a source of concentrated and 
injuriously hyperosmotic cryoprotectant solution which will  
be delivered undiluted to the patient. 

      The solution to this problem has been to vigorously 
stir the contents of the recirculating reservoir so that 
added cryoprotectant concentrate is quickly diluted and 
mixed with the recirculating perfusate. Mixing is typically 
achieved through the use of a teflon coated, magnetically 
driven stirring bar identical to those used to mix solutions 
both in the laboratory and in industry (See figure 2).

* Illustration not shown.

FIGURE 2: Typical magnetically driven laboratory stirrer 

      A consequence of the stirring of the recirculating 
reservoir by the rapidly spinning magnetic stir bar is the 
generation of an air vortex in the recirculating perfusate.  
While this vortex is very effective at both rapidly and 
completely mixing the concentrate with the perfusate in the 
recirculating reservoir, it is also very effective at 
introducing air into the recirculating perfusate as well. At 
rates of rotation fast enough to achieve good mixing, the 
bottom of the vortex of air reaches the rapidly rotating 
stir bar.  Air is thus turbulently mixed into the perfusate 
where it forms bubbles of widely varying size; the smallest 
of which are very stable.  As the concentration of 
cryoprotectant rises, and the viscosity of the solution 
correspondingly increases, air bubbles generated by stirring 
in the recirculating reservoir become more and more stable 
and begin to saturate the recirculating perfusate creating 
large amounts of foam.

      This phenomenon was first observed by the author 
during a human cryoprotective perfusion carried out at the 
Alcor Foundation in Riverside, California in August of 1991. 
During that case the top of the acrylic housing of the 
hollow fiber bundle of the Sarns  16310 oxygenator and the 
top half of the housing of the Pall EC-1440 40 micron 
extracorporeal filter were noted to contain large amounts of 
foam.  A careful examination of the arterial line leading 
from the filter to the patient did not disclose the presence 
of any visible bubbles, but the phenomenon was nevertheless 
very troubling and constituted an unacceptable hazard.

      Introduction of air into the arterial circulation 
during cardiopulmonary bypass (perfusion) is a catastrophe 
about which many articles have been written and about which 
many pages of any textbook dealing with perfusion will be 
dedicated (5).  Introduction of air into the arterial 
circulation leads to generation of emboli interrupting the 
flow of blood or perfusate to the tissues.  While 
extracorporeal filters are reasonably effective at excluding 
*limited amounts* of macroscopic air, they allow some 
microbubbles to pass.  Elimination of foam from the arterial 
side of the extracorporeal circuit is thus of paramount 
importance and constitutes a fundamental of safe and 
responsible perfusion of cryopreservation patients.

      Attempts to control the generation of foam as a result 
of stirring the recirculating reservoir initially consisted 
of keeping the perfusate level in the recirculating 
reservoir high and keeping the r.p.m. of the stirrer bar to 
the minimum required to achieve thorough mixing.

      While this approach was moderately successful in 
reducing the amount of foam generated during perfusion, it 
was not completely effective.  Furthermore, as the desired 
terminal patient cryoprotectant concentration (i.e., the 
desired terminal concentration of glycerol in the patient's 
tissues) has risen from 4M where it was in 1985 (6) to the 
currently recommended 6M (7) the problem of foam generation 
secondary to stirring of the recirculating reservoir has 
increased as a result of the increasing peak viscosity of 
the recirculating perfusate; perfusate containing 6M 
glycerol is far more viscous than perfusate containing 4M 
glycerol. More viscous perfusate results in more stable 

      Recently Biopreservation began a series of experiments 
wherein dogs are subjected to transport, blood washout and 
cryoprotective perfusion to 6M glycerol employing a model 
closely approximating that used to cryopreserve human 
patients.  During the course of the cryoprotective perfusion 
of two of these animals generation of significant amounts of 
foam was again observed despite efforts to minimize air 
entrainment by the stir bar.  While the Pall 40 micron 
extracorporeal filter appeared to trap most of this foam, 
microbubbles were detected ultrasonically using a Renal 
Systems Sonalarm Foam Detector.  Furthermore, at the 
conclusion of cryoprotective perfusion a fine line of 
microbubbles was observed in the arterial line leading from 
the Pall filter to the femoral arterial cannula.


      These observations lead to redoubled efforts to solve 
the problem of microbubble/foam generation during stirring 
of the recirculating perfusate.

      The first attempt to solve the problem was made by 
floating a polyethylene lid atop the liquid in the 
recirculating reservoir to prevent generation of an air 
vortex.  This lid consisted of a circular  3.5 cm high dish 
of polyethylene (closely resembling an inverted plastic tank 
lid) of slightly smaller diameter than the recirculating 
reservoir. The flat surface of the lid contained small 
raised air cells to entrap air and allow the lid to stably 
float. Unfortunately, the vigorous stirring required to mix 
viscous multimolar solutions of glycerol created sufficient 
turbulence at the top of the reservoir to result in tipping, 
filling and sinking/tumbling of the floating lid.  
Additionally, the air entrapped in the lid air cells was 
also entrained into the solution generating foam as a 
result.  It thus became clear that elimination of the air 
vortex would require a floating lid which could not be 
tipped by turbulent fluid flow and which was carefully 
designed to exclude all air/fluid contact.

      A second generation floating lid was then developed  
which consisted of a custom-fabricated hollow cylinder of 
high density polypropylene 11 cm high by 33 cm wide (See 
Figure 3)(manufactured by Custom Fab or Riverside, CA).  
This cylinder was closed at both the top and the bottom.  
The top surface of the lid was penetrated by a 5 cm screw-
cap port opening.  This top port was sealed by an 
unscrewable handle approximately 10 cm tall which could 
removed to allow for the addition of sterile solution to the 
lid so that the depth to which the lid sank in the 
recirculating perfusate could be controlled by moderating 
its buoyancy with added fluid ballast.  This was found to be 
of importance since allowing the lid to float too high 
permitted air to be entrained beneath the lid as it wobbled 
atop the turbulent column of recirculating perfusate.  This 
wobble could be greatly reduced both by partially sinking 
the lid in the perfusate, and by weighting it with the same 
fluid ballast use to partuially submerge it, thus 
effectively increasing the amount of energy required to 
disturb the lid. 

*Illustration not shown.

FIGURE 3: Variably bouyant floating lid assembly.

     While the use of this sealed, variably buoyant lid 
(VBL) was very effective at preventing air from being 
introduced if it was applied without interruption from the 
start of perfusion, it could not eliminate air that was 
accidentally introduced after perfusion began, such as 
introduction of air into the venous return line or into the 
cryoprotectant concentrate addition line.  While these 
sources of air are not likely to be routinely encountered, 
they nevertheless exist as real possibilities. Further, once 
air is introduced and converted into microbubbles, it is 
stable and difficult to get rid of.


      In order to deal with this problem a second series of 
experiments was conducted using both 1% human albumin in 
water and 4M glycerol with 2% human albumin in water in a 
recirculating system identical to that employed in human 
cryopreservations.  Both the albumin-water and the glycerol-
albumin-water solutions were very effective at generating 
large amounts of stable foam when stirred with a magnetic 
stir bar at rates comparable to those employed to mix the 
recirculating perfusate during human and canine 
cryoprotective perfusions.  While the use of the  (VBL) was 
very effective at preventing foam generation it did nothing 
to deal with the problem once it occurred.

      To solve the problem of foam generated in the 
recirculating reservoir as a result of the inadvertent 
introduction of air after the VBL was in place two changes 
to the system were made.  First, the VBL was treated with 
Dow -Corning Antifoam-A (Dow-Corning, Midland, MI)  so that 
foam accumulating under it would be decomposed into large 
bubbles which the centrifugal force of the rotating fluid 
column might more easily push out from under the VBL.  
Secondly, a Sarns 9438 Filtered Venous Reservoir was 
inserted in-line between the recirculating reservoir and the 
intake of the arterial pump.  The Sarns 8438 reservoir has a 
large .15 meter surface area 40 micron filter which is 
underlaid with a layer of Antifoam A-treated coarse 
debubbling material and overlaid with a layer of Antifoam-A-
treated fine debubbling material.  

      This reservoir is capable of being operated under 
vacuum without leaking air (it is designed to function as a 
cardiotomy reservoir as well) and consequently is suitable 
for use on the negative pressure side of the perfusion 
circuit.  The perfusate level in this reservoir was adjusted 
(and maintained) at the minimum level of 400 cc (reservoir 
capacity is 4500 cc) by use of a Mityvac hand-held vacuum 
pump manufactured by Neward Enterprises of Upland, 

      The interposition of the Sarns 9438 reservoir between 
the recirculating reservoir and the arterial pump was 
effective at removing all foam and bubbles introduced as a 
result of stirring the recirculating reservoir with or 
without the use of the VBL.  One added advantage to the use 
of the Sarns reservoir is the extra filtering capacity which 
is likely to be especially useful during perfusion of 
patients with intravascular clotting and/or cold 
agglutination where steady streams of particulate matter in 
the patient's venous return are encountered which might load 
the comparatively small surface area of the arterial filter.  

      This system, consisting of both the VBL and the Sarns 
9438 Reservoir, as shown in Figure 4, was then used during 
the cryoprotective perfusion of a dog to 6.5M glycerol.  The 
system was found to perform as effectively in a model 
simulating almost exactly the conditions encountered during 
cryoprotective perfusion of humans for long-term 
*Illustration not shown.

FIGURE 4: Human cryoprotective perfusion circuit 
incorporating VBL and Sarn 9438 reservoir.

      On 6 March, 1994 this system was employed clinically 
for the first time during the cryoprotective perfusion of 
American Cryonics Society patient ACS 9577.  The system 
functioned flawlessly and allowed perfusion of the patient 
to a terminal concentration of 6.76M glycerol without the 
introduction of any air into the arterial perfusate.  
Perfusate coming from the recirculating reservoir to the 
Sarns 9438 reservoir and from the Sarns reservoir to the 
oxygenator and extracorporeal filter (Pall EC-1440) was 
observed to be free of air.  The VBL was sunk approximately 
2 cm into the recirculating solution by the addition of 
approximately 2L of Dianeal 5% dextrose containing 
peritoneal dialysis solution to the lid as ballast. (Dianeal 
was chosen simply because it was cheap and available; we had 
a large overstock of it on hand.)


      An undesirable side effect of vigorous stirring of the 
recirculating perfusate reservoir during human 
cryoprotective perfusions is the introduction of air into 
the recirculating perfusate resulting in the generation of 
foam.  Elimination of this air is achieved by a two-step 
process: prevention of air entrainment by elimination of the 
air vortex in the recirculating reservoir through the use of 
a variably buoyant lid, and defoaming and filtration of the 
recirculating perfusate by interposition of a Sarns 8438 
Venous Filtration reservoir between the recirculating 
reservoir and the intake of the arterial pump.  This system 
has been shown to be effective at eliminating foam 
generation during both experimental and clinical 
cryoprotective perfusion.

1) Levin, RL. A generalized method for the minimization of 
cellular osmotic stresses and strains during the 
introduction and removal of permeable cryoprotective agents. 
J. Biomech. Eng. 1982;104:81-86.

2) Darwin, MG, Leaf, JD, and Hixon, HL. Case report: 
neuropreservation of Alcor patient A-1068. Cryonics 

3) Jaconsen, IA and Pegg, DE . Cryoprotection of rabbit 
kidneys with glycerol. in Organ Preservation II, eds. DE 
Pegg and IA Jacobsen, New York: Churchill Livingstone. 1979.

4)Perry RM. Mathematical analysis of recirculating perfusion 
systems with apoplication to cryonic suspension. Cryonics 

5) Reed, CC, Kurusz, M, and Lawrence, JR. EA. Safety And 
Techniques In Perfusion. Stafford, TX: Quali-Med, Inc. 1988.

6) Darwin, MG, Leaf, JD, and Hixon, HL, ibid.

7) Fahy, GM, personal communication.