X-Message-Number: 1917
From:  (Nick Szabo)
Subject: CRYONICS: Vitrification of complex embryos
Date: Mon, 8 Mar 1993 12:14:20 -0800 (PST)


[Science 258:1932, 1992]  Mazur et. al., "Cryobiological Preservation of
Drosophila Embryos" 

Abstract: 

The inability to cryobiologically preserve the fruit fly _Drosophila
melanogaster_ has  required that fly stocks be maintained by frequent 
transfer of adults.  This method is costly in terms of time and can 
lead to loss of stocks.  Traditional slow freezing methods do not 
succeed because the embryos are highly sensitive to chilling.
With the procedures described here, 68% of precisely staged 15-hour 
Oregon R (wild-type) embryos hatch after vitrification at -205C, and 
40% of the resulting larvae develop into normal adult flies.  These 
embryos are among the most complex organisms successfully preserved by 
cryobiology. 

Some relevant excerpts: 

_A priori_, one might consider embryos at the early stages better candidates 
for preservation than those at later stages because of their simpler
structure.  We and Steponkus and co-workers found independently, however, that
early embryonic stages of _Drosophila_ were far poorer candidates for
cryopreservation than 12-  to 13-hour embryos, even though the latter 
contain about 50,000 cells that are well differentiated into organ systems...
In contrast, mouse embryos are generally frozen at the 8-cell stage, and 
C. elegans larvae at the 558-cell stage. 

...the embryos are surrounded by a vitelline membrane that is rendered 
impermeable by waxes, and the larvae and adults possess a cuticle that 
is impregnated with lipids. To achieve cryobiological preservation, these 
barrier compounds must be removed without injury...our protocol exposes 
embryos for precise times (90 to 110 s) to heptane containing low (0.3 or 
0.4%) and precise concentrations of alchohol (c-butanol). 

The embryos are highly chill sensitive...if unpermeabilized 12-hour embryos 
are cooled at <1C per minute,nearly all of them are killed before reaching 
-25C.... at the usual concentrations of cryoprotectants (1 to 1.5M), cooling 
rates of 20,000C per minute would garuntee lethal intracellular freezing.
The only way to avoid such intracellular freezing is to introduce very high
concentrate (>50% wt) of glass-inducing solutes in and around the embryos,
concentrations that are high enough both to induce vitrification during 
cooling and to prevent devitrification (crystallization) during warming 
and yet not be toxic.  Steponkus et. al. reported that ethylene glycol 
can act as a glass-inducing solute when added in two steps in a manner 
similar to Rall's approach for mouse embryos.  In Steponkus'
procedure: (i) The embryos are first fully permeated by 2.1M ethylene 
glycol at 22C.  (ii) The intracellular concentration of ethylene glycol 
is then raised to high levels by osmotically dehydrating the embryos by 
a 5- to 8-minute exposure to 8.5M ethylene glycol at 0C.  After this 
dehydration step, the embryos are cooled at ~25,000C per minute to
-205C in nitrogen slush.  Upon rapid rewarming, 18% of the 13-hour embryos 
hatched; however, only 3% of the resulting larvae developed into adult flies. 

...We have found survival to be far more sensitive to the warming rate than 
to the cooling rate.  Thus, decreasing the warming rate from 100,000 to
2,000C per minute dropped survival from 12 to 0%, whereas a similar lowering 
of the cooling rate had much less effect.  This assymetry strongly suggests 
that chilling injury is less a contributor to loss in viability than is 
damage from the devitrification of critical portions of the vitrified 
cytoplasm during warming, probably because those compartments 
contain insufficient ethylene glycol.   
	
...with a developmentally more precise way of staging the embryos and 
modifications in the posttreatment culture techniques, 60 to 75% of 
vitrified embryos hatched, and a mean of 40% of the resulting larvae 
developed into fertile adults...[this efficiency of 25%] is similar to 
that obtained with mouse embryos cryobiologically preserved under optimal
conditions...the time the organism is maintained at -200C is irrelevant. 
 
[zero time = 12.5 hours of development] 
...The fraction [permeabilized by butanol-heptane] dropped from 90% at 
zero time to about 80% at +2.4 hours, the time yielding the peak hatching
survival of 68%.  By +3.9  hours, the percentage permeabilized dropped to 
58% and became more variable; the percentage hatching dropped to 10%.  
After another hour of development, both the percentage of embryos that 
become permeabilized and the percentage that survive cryogenic exposure 
drop to or near zero...we interpret these data to mean that portions of 
the cuticle begin to become refractory to permeabilization by butanol-heptane
between +1.5 and +2.5 (14 and 15) hours of development. Consequently, 
compartments in the embryo do not attain sufficiently high quantities 
of ethylene glycol to maintain the vitreous state during cooling and warming. 

...[the percentage of hatching larvae that develop into adults] curve 
parallels the hatching curve...the factors that cause hatching percentages 
to be low inflict damage on most larvae, and such damage prevents subsequent
development to adulthood. 

...the major developmental processes are completed by stage 16 [where optimal 
hatching rate occurs]...we believe our findings have more general implications 
for cryobiology.  The optimal development stages being frozen are probably 
the most complex systems that have been cryobiologically preserved.  The 
embryos are highly differentiated into tissues and organs including muscle 
and nerve, which indicates that differentiated multicellularity is not a 
barrier to cryopreservrification procedures are required to obtain survival... 

Nick Szabo					 

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